Nuclear receptor ligands and ligand binding domains

ABSTRACT

The present invention provides new methods, particularly computational methods, and compositions for the generation of nuclear receptor synthetic ligands based on the three dimensional structure of nuclear receptors, particularly the thyroid receptor (herein referred to as “TR”). Also provided are crystals, nuclear receptor synthetic ligands, and related methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following provisionalapplications: U.S. Ser. No. 60/008,540 and 60/008,543, filed Dec. 13,1995, and Ser. No. 60/008,606, filed Dec. 14, 1995. This applicationclaims the benefit of the following U.S. patent application: U.S. Ser.No. 08/764,870, filed Dec. 13, 1996.

ACKNOWLEDGMENTS

This invention was supported in part by grants from the NationalInstitutes of Health grant number 1 R01 DK 43787 and 5 R01 DK 41842. TheU.S. Government may have certain rights in this invention.

INTRODUCTION

1. Technical Field

This invention relates to computational methods for designing ligandsthat bind to nuclear receptors, crystals of nuclear receptors, syntheticligands of nuclear receptors and methods of using synthetic ligands.

2. Background

Nuclear receptors represent a superfamily of proteins that specificallybind a physiologically relevant small molecule, such as hormone orvitamin. As a result of a molecule binding to a nuclear receptor, thenuclear receptor changes the ability of a cell to transcribe DNA, i.e.nuclear receptors modulate the transcription of DNA, although they mayhave transcription independent actions. Unlike integral membranereceptors and membrane associated receptors, the nuclear receptorsreside in either the cytoplasm or nucleus of eukaryotic cells. Thus,nuclear receptors comprise a class of intracellular, solubleligand-regulated transcription factors.

Nuclear receptors include receptors for glucocorticoids (GRs), androgens(ARs), mineralocorticoids (MRs), progestins (PRs), estrogens (ERs),thyroid hormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs),peroxisomes (XPARs and PPARs) and icosanoids (IRs). The so called“orphan receptors” are also part of the nuclear receptor superfamily, asthey are structurally homologous to the classic nuclear receptors, suchas steroid and thyroid receptors. To date, ligands have not beenidentified with orphan receptors but it is likely that small moleculeligands will be discovered in the near future for this class oftranscription factors. Generally, nuclear receptors specifically bindphysiologically relevant small molecules with high affinity and apparentKd's are commonly in the 0.01-20 nM range, depending on the nuclearreceptor/ligand pair.

Development of synthetic ligands that specifically bind to nuclearreceptors has been largely guided by the trial and error method of drugdesign despite the importance of nuclear receptors in a myriad ofphysiological processes and medical conditions such as hypertension,inflammation, hormone dependent cancers (e.g. breast and prostatecancer), modulation of reproductive organ function, hyperthyroidism,hypercholesterolemia and obesity. Previously, new ligands specific fornuclear receptors were discovered in the absence of information on thethree dimensional structure of a nuclear receptor with a bound ligand.Before the present invention, researchers were essentially discoveringnuclear receptor ligands by probing in the dark and without the abilityto visualize how the amino acids of a nuclear receptor held a ligand inits grasp.

Consequently, it would be advantageous to devise methods andcompositions for reducing the time required to discover ligands tonuclear receptors, synthesize such compounds and administer suchcompounds to organisms to modulate physiological processes regulated bynuclear receptors.

SUMMARY OF THE INVENTION

The present invention provides for crystals of nuclear receptor ligandbinding domains with a ligand bound to the ligand binding domain (LBD).The crystals of the present invention provide excellent atomicresolution of the amino acids that interact with nuclear receptorligand, especially thyroid receptor ligands. The three dimensional modelof a nuclear receptor LBD with a ligand bound reveals a previouslyunknown structure for nuclear receptors and shows that the ligand isbound in a water inaccessible binding cavity of the ligand bindingdomain of the nuclear receptor.

The present invention also provides for computational methods usingthree dimensional models of nuclear receptors that are based on crystalsof nuclear receptor LBDs. Generally, the computational method ofdesigning a nuclear receptor ligand determines which amino acid or aminoacids of a nuclear receptor LBD interact with a chemical moiety (atleast one) of the ligand using a three dimensional model of acrystallized protein comprising a nuclear receptor LBD with a boundligand, and selecting a chemical modification (at least one) of thechemical moiety to produce a second chemical moiety with a structurethat either decreases or increases an interaction between theinteracting amino acid and the second chemical moiety compared to theinteraction between the interacting amino acid and the correspondingchemical moiety on the natural hormone.

Also provided is a method of modulating the activity of a nuclearreceptor. The method can be in vitro or in vivo. The method comprisesadministering in vitro or in vivo a sufficient amount of a compound ofthe following formula:

where the compound fits specially and preferentially into a nuclearhormone receptor LBD of interest. The method is exemplified bymodulating the activity of a thyroid receptor (TR). For modulating TRactivity, a compound of Formula I is employed that fits spacially andpreferentially into a TR ligand binding domain (TR LBD), includingcompounds specific for a TR LBD isoform of interest. Of particularinterest are the TR LBD isoforms α (TR-α) and β (TR-β). Additionalcompounds of interest include derivatives of Formula I, such as thosecompounds having the biphenyl (φ-X-φ) or single phenyl (φ-X or X-φ)nucleus of Formula I and its corresponding substituent groups describedherein. Compounds that are interatively designed using structuralinformation gleaned from these compounds and which modulate nuclearhormone receptor activity also are of interest.

The present invention also includes a method for identifying a compoundcapable of selectively modulating the activity of a nuclear receptor.This aspect of the invention is exemplified by a method for identifyinga compound capable of selectively modulating the activity of a TRisoform. The method comprises modeling test compounds that fit spaciallyand preferentially into a TR LBD isoform of interest using an atomicstructural model of a TR LBD isoform bound to a test compound, screeningthe test compounds in a biological assay for TR isoform activitycharacterized by binding of a test compound to a TR LBD isoform, andidentifying a test compound that selectively modulates the activity of aTR isoform. The compounds may be those of Formula I or derivativesthereof, including compounds having a biphenyl or single phenyl nucleusof Formula I.

Further included is a method for identifying agonist or antagonistligands of a nuclear receptor using the atomic coordinates of a LBD inconjunction with a computerized modeling system. This aspect of theinvention is exemplified by identifying a TR agonist or antagonistligand by providing the atomic coordinates of a TR LBD to a computerizedmodeling system, modeling ligands which fit spacially into the TR LBD,and identifying in a biological assay for TR activity a ligand whichincreases or decreases TR activity. The compounds can be those ofFormula I or derivatives thereof, including compounds having a biphenylor single phenyl nucleus of Formula I.

Also provided is a method of identifying a compound that selectivelymodulates the activity of one type of nuclear receptor compared to othernuclear hormone receptors. The method is exemplified by modeling testcompounds which fit spacially into a TR LBD using an atomic structuralmodel of a TR LBD, selecting a compound comprising conformationallyconstrained structural features that interact with conformationallyconstrained residues of a TR LBD, and identifying in a biological assayfor TR activity a compound that selectively binds to a TR LBD comparedto other nuclear receptors. The conformationally constrained featuresinvolved in receptor-selective ligand binding can be identified bycomparing atomic models of receptor isoforms bound to the same and/ordifferent ligands. The methods facilitate design and selection ofcompounds that have increased selectivity for a particular nuclearreceptor. The compounds may be those of Formula I or derivativesthereof, including compounds having the biphenyl or single phenylnucleus of Formula I.

Another aspect of the invention is a method for increasing the receptorselectivity of a compound for a particular type of nuclear receptor.This involves the chemical modification of a substituent group of acompound of Formula I to generate compounds which have increasedselectivity for one type of receptor. For example, chemical modificationof a substituent group of the compound of Formula I can be used tointroduce additional constraints into a compound that modulates TRactivity to increase its selectivity in vivo for TR-type receptors.Additional constraints also may be added for stability. The modifiedgroups will preferably interact with a conformationally constrainedstructural feature of a TR LBD that is conserved among TR isoforms. Amore preferred method comprises selecting compounds havingconformationally constrained groups that interact with conformationallyconstrained residues of a TR LBD conserved among TR isoforms. Thecompounds can be those of Formula I or derivatives thereof, includingcompounds having the biphenyl or single phenyl nucleus of Formula I.

The invention finds use in the selection and characterization ofpeptide, peptidomimetic or synthetic compounds identified by the methodsof the invention, particularly new lead compounds useful in treatingdisorders related to nuclear receptor-based deficiencies, includingTR-related disorders. For TR-related disorders, the compounds andmethods of the invention can be used to modulate TR activity byadministering to a mammal in need thereof a sufficient amount ofcompound of Formula I or derivative thereof that fits spacially andpreferentially into a TR LBD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating computational methods for designingligands that interact with nuclear receptors of the nuclear receptorsuperfamily.

FIG. 2 is a schematic representation of nuclear receptor structures,indicating regions of homology within family members and functions ofthe various domains.

FIG. 3 shows the aligned amino acid sequences of the ligand bindingdomains of several members of the nuclear receptor superfamily.

FIG. 4 is a ribbon drawing of the rat TR-α LBD with secondary structureelements labelled. The ligand (magenta) is depicted as a space-fillingmodel. Alpha helices and coil conformations are yellow, beta strands areblue.

FIG. 5 shows two cross-sections of a space-filling model of rat TR-αexposing the ligand (magenta) tightly packed within the receptor.

FIG. 6 is a schematic of the ligand binding cavity. Residues whichinteract with the ligand appear approximately at the site ofinteraction. Hydrogen bonds are shown as dashed lines between thebonding partners; distances for each bond are listed. Non-bondedcontacts are shown as radial spokes which face toward interacting atoms.

FIG. 7 is the distribution of crystallographic temperature factors inthe refined rat TR-α LBD. The distribution is represented as a colorgradation ranging from less than 15 (dark blue) to greater than 35(yellow-green).

FIG. 8 is a ribbon drawing of the rat TR-α LBD showing the c-terminalactivation domain to ligand. Residues which comprise the c-terminalactivation domain (Pro393-Phe405) are depicted as a stickrepresentation. Hydrophobic residues, particularly Phe401 and Phe405(blue) face inwards toward the ligand. Glu403 (red) projects outwardinto the solvent.

FIG. 9 is an electrostatic potential surface of the rat TR-α LBD,calculated using GRAPH. Negative electrostatic potential is red;positive electrostatic potential is blue. The c-terminal activationdomain forms a largely hydrophobic (white). The Glu403 is presented as asingular patch of negative charge (red).

FIG. 10 is a diagram comparing agonists and antagonists for severalnuclear receptors.

FIG. 11 is the synthetic scheme for preparation of TS1, TS2, TS3, TS4and TS5.

FIG. 12 is the synthetic scheme for preparation of TS6 and TS7.

FIG. 13 is the synthetic scheme for preparation of TS8.

FIG. 14 is the synthetic scheme for preparation of TS10.

FIG. 15 depicts the chemical structures of several TR ligands.

FIG. 16 is a graph illustrating competition assays in which T₃ and Triaccompete with labeled T₃ for binding to human TR-α or human TR-β.

FIG. 17 depicts a Scatchard analysis of labelled T₃ binding to TR-α andTR-β.

FIG. 18 is a chart showing the effect of TS-10 on the transcriptionalregulation of the DR4-ALP reporter gene in the presence or absence of T3as assayed in TRAFα1 reporter cells.

FIG. 19 is a chart showing the effect of TS-10 on the transcriptionalregulation of the DR4-ALP reporter gene in the presence or absence of T3as assayed in TRAFβ1 reporter cells.

FIG. 20 is a chart showing the effect of TS-10 on the transcriptionalregulation of the DR4-ALP reporter gene in the presence or absence of T3as assayed in HepG2, a liver reporter cell line.

FIG. 21 is a partial ribbon drawing of TR-α LBD with T3 in the ligandbinding cavity. Selected interacting amino acids are labelled, includingIle221, Ile222 and Ser260, Ala263, Ile299 and Leu 276.

FIG. 22 is a partial ribbon drawing of TR-α LBD with T3 and Dimitsuperimposed in the ligand binding cavity. Interactions with Ile221,Ile222, Ala260, Ile 299 and Leu276 are labelled.

FIG. 23 is a partial ribbon drawing of TR-α LBD with T3, illustratingthe three Arginine residues (Arg228, Arg262 and Arg 266 (dark stickfigures)) of the polar pocket, three water molecules HOH502, HOH503 andHOH504, with hydrogen bonds indicated by dotted lines.

FIG. 24 is a partial ribbon drawing of TR-α LBD with Triac, illustratingthe three Arginine residues (dark stick figures) of the polar pocket,water molecules (HOH503, HOH504 and HOH600), with hydrogen bondsindicated by dotted lines.

FIG. 25 is a partial ribbon drawing of the TR-α LBD with T3 and Triacsuperimposed in the ligand binding cavity. The drawing shows severalinteracting amino acid residues in the polar pocket that remainunchanged whether T3 or Triac occupies the ligand binding cavity:Arg262, Asn179, HOH503 and HOH504, and Ser277. Both Arg228 and Arg 266occupy two different positions, depending on whether T3 or Triac isbound.

FIGS. 26A and 26B are stereochemical representations of the TR-α LBDwith Dimit bound.

FIG. 27 is a partial ribbon drawing of TR-β LBD with GC-1 in the ligandbinding cavity. Amino acids Arg282, Arg316, Arg320, Asn 331 and His435are labelled.

FIG. 28 is a partial ribbon drawing of TR-β LBD with Triac in the ligandbinding cavity. Amino acids Arg282, Arg316, Arg320, Asn331 and His435are labelled.

FIG. 29 is a partial ribbon drawing of TR-βLBD with GC-1 (Blue)overlayed with TR-α LBD with Dimit (Red) in the ligand binding cavities.Amino acids Arg228, Arg262, Arg266 and Ser277 (TR-α LBD), and Arg282,Arg316, Arg320 and Asn331 (TR-β LBD) are labelled.

FIG. 30 is a partial ribbon drawing of TR-β LBD with Triac (Blue)overlayed with TR-α LBD with Triac (Red) in the ligand binding cavities.Amino acids Arg228, Arg262, Arg266, Ser277 and His381 (TR-α LBD), andArg282, Arg316, Arg320 and His435 (TR-β LBD) are labelled.

FIG. 31 is a graph showing competition curves comparing wildtype TR-αand TR-β to a variant TR-β having a single amino acid substitution inthe ligand binding domain.

FIG. 32 shows atomic numbering for thyronine-like ligands.

APPENDIX 1 is an appendix of references.

APPENDIX 2 is a chart of amino acids that interact with a TR ligand, forTR complexed with Dimit, Triac, IpBr2, T3 and GC-1.

APPENDIX 3 is a chart of atomic coordinates for the crystal of rat TR-αLBD complexed with Dimit.

APPENDIX 4 is a chart of atomic coordinates for the crystal of rat TR-αLBD complexed with Triac.

APPENDIX 5 is a chart of atomic coordinates for the crystal of rat TR-αLBD complexed with IpBr₂.

APPENDIX 6 is a chart of atomic coordinates for the crystal of rat TR-αLBD complexed with T₃.

APPENDIX 7 is a chart of atomic coordinates for the crystal of humanTR-β LBD complexed with Triac.

APPENDIX 8 is a chart of atomic coordinates for the crystal of humanTR-β-LBD complexed with GC-1.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention provides new methods, particularly computationalmethods, and compositions for the generation of nuclear receptorsynthetic ligands based on the three dimensional structure of nuclearreceptors, particularly the thyroid receptor (herein referred to as“TR”). Previously, the lack of three dimensional structural informationabout the ligand binding domain of a nuclear receptor thwarted the fieldof nuclear receptor drug discovery, especially the absence of threedimensional structural information relating to a nuclear receptor with aligand bound.

Described herein for the first time are crystals and three dimensionalstructural information from a nuclear receptor's ligand binding domain(LBD) with a ligand bound. The structure of the TR LBD complexed with3,5,3′-triiodothyronine (T₃), 3,5-dibromo-3′-isopropylthyronine (IpBr2),3,5-dimethyl-3′-isopropylthyronine (Dimit), and3,5,3′-triiodothyroacetic acid (Triac),3,5-dimethyl-4-(4′-hydroxy-3′isopropylbenzyl)-phenoxy acetic acid (GC1)are exemplified. Such crystals offer superior resolution at the atomiclevel and the ability to visualize the coordination of nuclear receptorligands by amino acids that comprise the LBD. The present invention alsoprovides computational methods for designing nuclear receptor syntheticligands using such crystal and three dimensional structural informationto generate synthetic ligands that modulate the conformational changesof a nuclear receptor's LBD. Such synthetic ligands can be designedusing the computational methods described herein and shown, in part, inFIG. 1. These computational methods are particularly useful in designingan antagonist or partial agonist to a nuclear receptor, wherein theantagonist or partial agonist has an extended moiety that prevents anyone of a number of ligand-induced molecular events that alter thereceptor's influence on the regulation of gene expression, such aspreventing the normal coordination of the activation domain observed fora naturally occurring ligand or other ligands that mimic the naturallyoccurring ligand, such as an agonist. As described herein, syntheticligands of nuclear receptors will be useful in modulating nuclearreceptor activity in a variety of medical conditions.

Of particular interest is use of such ligands in a method of modulatingTR activity in a mammal by administering to a mammal in need thereof asufficient amount of a compound of Formula I,

where the compound fits spatially and preferentially into a TR LBD. By“fits spacially” is intended that the three-dimensional structure of acompound is accommodated geometrically by a cavity or pocket of a TRLBD. By “TR LBD” is intended a structural segment or segments of thyroidhormone receptor polypeptide chain folded in such a way so as to givethe proper geometry and amino acid residue configuration for ligandbinding. This is the physical arrangement of protein atoms inthree-dimensional space forming a ligand binding pocket or cavity. By“fits spacially and preferentially” is intended that a compoundpossesses a three-dimensional structure and conformation for selectivelyinteracting with a TR LBD. Compounds of interest also includederivatives of Formula I. By “derivatives of Formula I” is intendedcompounds that comprise at least a single phenyl scaffold (φ-X or X-φ ofthe biphenyl scaffold (φX-φ) of Formula I which comprise thecorresponding substituents of Formula I desccribed herein. Compoundsthat are interatively designed using structural information gleaned fromthese compounds and which modulate nuclear hormone receptor activityalso are of interest. Preferred compounds of Formula I and itsderivatives that fit spacially and preferentially into a TR LBD comprisethe following substituents:

(i) an R1-substituent comprising an anionic group that interacts with aside chain nitrogen atom of an arginine corresponding to a residue fromthe group Arg228, Arg262, and Arg266 of human TR-α, and Arg282, Arg316and Arg320 of human TR-β, where the anionic group is about 1.7-4.0 Åfrom the nitrogen atom;

(ii) an R2-substituent comprising a hydrophobic or hydrophilic groupthat fits spacially into the TR LBD;

(iii) an R3-substituent comprising a hydrophobic or hydrophilic groupthat interacts with a side chain atom of a serine, alanine and/orisoleucine corresponding to a residue from the group Ser260, Ala263 andIle299 of human TR-α, and Ser314, Ala317 and Ile352 of human TR-β, wherethe hydrophobic or hydrophilic group is about 1.7-4.0 Å from the sidechain atom;

(iv) an R5-substituent comprising a hydrophobic or hydrophilic groupthat interacts with a side chain atom of a phenylalanine and/orisoleucine corresponding to a residue from the group Phe218, Ile221 andIle222 of human TR-α, and Phe272, Ile275 and Ile276 of human TR-β, wherethe hydrophobic or hydrophilic group is about 1.7-4.0 Å from the sidechain atom;

(v) an R6-substitutent comprising a hydrophobic or hydrophilic groupthat fits spacially into the TR LBD;

(vi) an X-substituent comprising a hydrophobic or hydrophilic group thatinteracts with a side chain atom of a leucine corresponding to a residuefrom the group Leu276 and Leu292 of human TR-α, and Leu 330 and Leu346of human TR-β, where the hydrophobic or hydrophilic group is about1.7-4.0 Å from the side chain atom;

(vii) an R2′-substituent comprising a hydrophobic or hydrophilic groupthat fits spacially into the TR LBD;

(viii) an R3′-substituent comprising a hydrophobic group that interactswith a side chain atom of a phenylalanine, glycine and/or methioninecorresponding to a residue from the group Phe215, Gly290, and Met388 ofhuman TR-α, and Phe269, Gly344, Met442 of human TR-β, where thehydrophobic group is about 1.7-4.0 Å from the side chain atom;

(ix) an R4′-substituent comprising an hydrogen bond donor or acceptorgroup that interacts with a side chain carbon or nitrogen atom of ahistidine corresponding to residue His381 of human TR-α, and His435 ofhuman TR-β, where the hydrogen bond donor or acceptor group is about1.7-4.0 Å from the side chain atom;

(x) an R5′-substituent comprising a hydrophobic or hydrophilic groupthat fits spacially into the TR LBD;

(xi) and R6′-substituent comprising a hydrophobic or hydrophilic groupthat fits spacially into the TR LBD; and

where the compound is other than thyronine (T3), triiodothyronine (T4)or other thyronine-like compounds previously known and used in a TRtreatment method, such as those referenced in Appendix I.

Examples of such substituents include the following:

where R₁ is

-   -   —O—CH₂CO₂H, —NHCH₂CO₂H, —CO₂H, —CH₂CO₂H, —CH₂CH₂CO₂H,        —CH₂CH₂CH₂CO₂H, —CH₂CH(NH₂CO₂H, —CH₂CH[NHCOCHφ₂]CO₂H,        —CH₂CH[NHCO(CH₂)₁₅CH₃]CO₂H, —CH₂CH[NH—FMOC]CO₂H,        —CH₂CH[NH-tBOC]CO₂H, or a carboxylate connected to the ring with        a 0 to 3 carbon linker,    -   —PO₃H₂, —CH₂PO₃H₂, —CH₂CH₂PO₃H₂, —CH₂CHNH₂PO₃H₂,        —CH₂CH[NHCOCHφ₂]PO₃H₂, —CH₂CH[NHCO(CH₂)₁₅CH₃]PO₃H₂,        —CH₂CH[NH—FMOC]PO₃H₂, —CH₂CH[NH-tBOC]PO₃H₂, or a phosphate or        phosphonate connected to the ring with a 0 to 3 carbon linker,    -   —SO₃H, —CH₂SO₃H, —CH₂CH₂SO₃H, —CH₂CHNH₂SO₃H,        —CH₂CH[NHCOCHφ₂]SO₃H, —CH₂CH[NHCO(CH₂)₁₅CH₃]SO₃H,        —CH₂CH[NH—FMOC]SO₃H, —CH₂CH[NH-tBOC]SO3H, or a sulfate or        sulfite connected to the ring with a 0 to 3 carbon linker, or        acts as the functional equivalent of CH₂CH(NH₂)CO₂H of T3 in the        molecular recognition domain when bound to a TR, wherein R₁ can        be optionally substituted with an amine,        where R₂ is    -   H, halogen, CF₃, OH, NH₂, SH, CH₃, -Et, or acts as the        functional equivalent of H in the molecular recognition domain        when bound to a TR,        where R₃ is    -   —H, halogen, —CF₃, —OH, —NH₂, —N₃, —SH, —CH₃, -Et, or acts as        the functional equivalent of I in the molecular recognition        domain when bound to a TR,        where R₅ is    -   —H, halogen, —CF₃, —OH, —NH₂, —N₃, —SH, —CH₃, -Et, or acts as        the functional equivalent of I in the molecular recognition        domain when bound to a TR, and R₃ can be identical to R₅,        where R₆ is    -   —H, halogen, —CF₃, —OH, —NH₂, —SH, —CH₃, or acts as the        functional equivalent of H in the molecular recognition domain        when bound to a TR, and R₂ can be identical to R₆,        where R₂′ is    -   —H, halogen, —CF₃, —OH, —NH₂, —N₃, —SH, —CH₃, -Et, or acts as        the functional equivalent of H in the molecular recognition        domain when bound to a TR,        where R₃′ is any hydrophobic group, including    -   halogen, —CF₃, —SH, alkyl, aryl, 5- or 6-membered heterocyclie,        cyano, or acts as the functional equivalent of I in the        molecular recognition domain when bound to a TR,        where R₄′ is    -   —H, halogen, —CF₃, —OH, —NH₂, NH₃, —N(CH₃)₃, carboxylate,        phosphonate, phosphate or sulfate, —SH, —CH₃, -Et, or akyl, aryl        or 5- or 6-membered heterocyclic aromatic attached through urea        or carbamate linkages to O or N or S at the R₄′ position, or        acts as the functional equivalent of OH in the molecular        recognition domain when bound to a TR,        where R₅′ is    -   —H, —OH, —NH₂, —N(CH₃)₂—SH—NH₃, —N(CH₃)₃, carboxylate,        phosphonate, phosphate, sulfate, branched or straight chain        alkyl having 1 to 9 carbons, substituted or unsubstituted aryl,        wherein said substituted aryl is substituted with halogen or 1        to 5 carbon alkyl and wherein said aryl is optionally connected        to the ring by a —CH₂—, aromatic heterocycle having 5 to 6        atoms, wherein said heterocycle may be substituted with one or        more groups selected from —OH, —NH₂, —SH, —NH₃, —N(CH₃)₃,        carboxylate, phosphonate, phosphate or sulfate, heteroalkyl,        arylalkyl, heteroaryl alkyl, polyaromatic, or        polyheteroaromatic, wherein said R₅′ may be substituted with        polar or charged groups,        where R₆′ is    -   —H, halogen, —CF₃, —OH, —NH₂, —SH, —CH₃, -Et, or acts as the        functional equivalent of H in the molecular recognition domain        when bound to a TR,        where X is    -   O, S, SO₂, NH, NR₇, CH₂, CHR₇, CR₇R₇, wherein R₇ is alkyl, aryl        or 5- or 6-membered heterocyclic aromatic,        and where the TR LBD ligand has an apparent Kd for binding TR        LBD of 1 μM or less.

Of particular interest are the class of compounds according to Formula Ihaving the following substituents: where R₁ is carboxylate, phosphonate,phosphate or sulfite and is connected to the ring with a 0 to 3 carbonlinker, R₂ is H, R₃ is —I, —Br, or —CH₃, R₅ is —I, —Br, or —CH₃, R₆ isH, R₂′ is H, R₃′ is —I, —Br, —CH₃, -iPr, -phenyl, benzyl, or 5- or6-membered ring heterocycles, R₄′ is —OH, —NH₂, and —SH, R₅′ is —H, —OH,—NH₂, —N(CH₃)₂—SH—NH₃, —N(CH₃)₃, carboxylate, phosphonate, phosphate,sulfate, branched or straight chain alkyl having 1 to 9 carbons,substituted or unsubstituted aryl, wherein said substituted aryl issubstituted with halogen or 1 to 5 carbon alkyl and wherein said aryl isoptionally connected to the ring by a —CH₂—, aromatic heterocycle having5 to 6 atoms, wherein said heterocycle may be substituted with one ormore groups selected from —OH, —NH₂, —SH, —NH₃, —N(CH₃)₃, carboxylate,phosphonate, phosphate or sulfate, heteroalkyl, arylalkyl, heteroarylalkyl, polyaromatic, or polyheteroaromatic, wherein said R₅′ may besubstituted with polar or charged groups, and R₆′ is H.

The present invention also includes a method for identifying a compoundcapable of selectively modulating the activity of a TR isoform. By“modulating” is intended increasing or decreasing activity of a TR. By“TR isoform” is intended TR proteins encoded by subtype and variant TRgenes. This includes TR-α and TR-β isoforms encoded by different genes(e.g., thra and thrb) and variants of the same genes (e.g., thrb1 andthrb2). The method comprises the steps of modeling test compounds thatfit spacially and preferentially into a TR LBD isoform of interest usingan atomic structural model of a TR LBD isoform bound to a test compound,screening the test compounds in a biological assay for TR isoformactivity characterized by binding of a test compound to a TR LBDisoform, and identifying a test compound that selectively modulates theactivity of a TR isoform. By “modeling” is intended quantitative andqualitative analysis of receptor-ligand structure/function based onthree-dimensional structural information and receptor-ligand interactionmodels. This includes conventional numeric-based molecular dynamic andenergy minimization models, interactive computer graphic models,modified molecular mechanics models, distance geometry and otherstructure-based constraint models. Modeling is preferably performedusing a computer and may be further optimized using known methods.

For selectively modulating activity of a TR isoform, such as TR-α orTR-β, a sufficient amount of a compound that fits spatially andpreferentially into TR LBD isoform is provided in vitro or in vivo toachieve the desired end result. TR-α isoform selectivity can beaccomplished with a compound comprising an anionic group that interactswith an oxygen or carbon of a serine residue corresponding to Ser277 ofhuman TR-α, where the anionic group is about 1.7-4.0 Å from the sidechain atom. TR-β isoform selectivity can be accomplished with a compoundcomprising an anionic group that interacts with the side chain nitrogenof an asparagine corresponding to Asn331 of human TR-β, where theanionic group is about 1.7-4.0 Å from the side chain nitrogen atom.

The present invention further includes a method for identifying a TRagonist or antagonist ligand by providing the atomic coordinates of a TRLBD to a computerized modeling system, modeling ligands which fitspacially into the TR LBD, and identifying in a biological assay for TRactivity a ligand which increases or decreases the activity of the TR.

The invention also involves a method for increasing receptor selectivityof a compound of Formula I or derivatives thereof for a TR-type receptorversus other nuclear receptors by selecting a compound that interactswith conformationally constrained residues of a TR LBD that areconserved among TR isoforms. “Conformationally constrained” is intendedto refer to the three-dimensional structure of a chemical or moietythereof having certain rotations about its bonds fixed by various localgeometric and physical-chemical constraints. In designing and selectingcompounds having increased specificity for TRs compared to other nuclearreceptors, the following methods of the invention can be used. Onemethod involves comparing atomic models of a first TR LBD isoform boundto a compound with a second TR LBD isoform bound to the same compound,identifying atoms of the TR LBD and compounds which interact, anddesigning or selecting a compound that interacts with TR LBD residuescomprising a conformationally constrained structural feature that isconserved between the TR LBD isoforms. Another method relates tocomparing a first TR LBD complexed with a first compound to a second TRLBD complexed with a second compound having one or more differentsubstituents compared to the first compound, identifying atoms of the TRLBD and compounds which interact, and designing or selecting compoundsthat interact with TR LBD residues comprising a conformationallyconstrained structural feature that is conserved between the TR LBDisoforms. The methods also facilitate identification of structural andconformationally constrained interactions that are conserved betweencompounds that bind to a TR LBD. The methods are exemplified bycomparing atomic models of a first TR LBD isoform complexed with a firstcompound of Formula I to a second TR LBD isoform complexed with thefirst compound, or a second compound of Formula I having differentsubstituents than the first compound. For example, a TR-α LBD bound to anatural hormone such as T3 is compared to a TR-β LBD bound to an organicthyronine-like compound such as GC-1. Conserved contacts are identifiedwhich are made between atoms of the different compounds and atoms of theTR LBDs, and the fiducial and adjustable components identified.Compounds selective for TR are identified in a biological assay for TRactivity that assays for selective binding to a TR and/or TRLBD-compared to other nuclear receptors. Conventional assays for TR andother nuclear receptors may be conducted in parallel or serially,including those assays described herein. Automatable methods arepreferred. The methods facilitate design and selection of compoundscomprising cyclic carbon and substituent atoms that interact with aconstrained side chain and/or main chain atom of a TR LBD residue.

In another aspect of the invention, the methods described herein areuseful for selecting peptides, peptidomimetics or synthetic moleculesthat modulate TR activity. Methods of the invention also find use incharacterizing structure/function relationships of natural and syntheticTR-ligands. Molecules of particular interest are new thyronine-likecompounds other than T3, T4 and other thyronine-like compoundspreviously known and used for treating TR-related disorders. Newcompounds of the invention include those which bind to a TR LBD isoformwith greater affinity than T3 or T4 and those which exhibitisoform-specific binding affinity.

Applicability to Nuclear Receptors

The present invention, particularly the computational methods, can beused to design drugs for a variety of nuclear receptors, such asreceptors for glucocorticoids (GRs), androgens (ARs), mineralocorticoids(MRs), progestins (PRs), estrogens (ERs), thyroid hormones (TRs),vitamin D (VDRs), retinoid (RARs and RXRs), icosanoid (IRs), andperoxisomes (XPARS and peroxisomal proliferators (PPAP)). The presentinvention can also be applied to the “orphan receptors,” as they arestructurally homologous in terms of modular domains and primarystructure to classic nuclear receptors, such as steroid and thyroidreceptors. The amino acid homologies of orphan receptors with othernuclear receptors ranges from very low (<15%) to in the range of 35%when compared to rat RARα and human TR-β receptors, for example. Inaddition, as is revealed by the X-ray crystallographic structure of theTR and structural analysis disclosed herein, the overall folding ofliganded superfamily members is likely to be similar. Although ligandshave not been identified with orphan receptors, once such ligands areidentified one skilled in the art will be able to apply the presentinvention to the design and use of such ligands, as their overallstructural modular motif will be similar to other nuclear receptorsdescribed herein.

Modular Functional Domains of Nuclear Receptors

The present invention will usually be applicable to all nuclearreceptors, as discussed herein, in part, to the patterns of nuclearreceptor activation, structure and modulation that have emerged as aconsequence of determining the three dimensional structures of nuclearreceptors with different ligands bound, notably the three dimensionalstructures or crystallized protein structure of the ligand bindingdomains for TR-α and TR-β. Proteins of the nuclear receptor superfamilydisplay substantial regions of amino acid homology, as described hereinand known in the art see FIG. 2. Members of this family display anoverall structural motif of three modular domains (which is similar tothe TR three modular domain motif):

1) a variable amino-terminal domain;

2) a highly conserved DNA-binding domain (DBD); and

3) a less conserved carboxyl-terminal LBD.

The modularity of this superfamily permits different domains of eachprotein to separately accomplish different functions, although thedomains can influence each other. The separate function of a domain isusually preserved when a particular domain is isolated from theremainder of the protein. Using conventional protein chemistrytechniques a modular domain can sometimes be separated from the parentprotein. Using conventional molecular biology techniques each domain canusually be separately expressed with its original function intact orchimerics of two different nuclear receptors can be constructed, whereinthe chimerics retain the properties of the individual functional domainsof the respective nuclear receptors from which the chimerics weregenerated.

FIG. 2 provides a schematic representation of family member structures,indicating regions of homology within family members and functions ofthe various domains.

Amino Terminal Domain

The amino terminal domain is the least conserved of the three domainsand varies markedly in size among nuclear receptor superfamily members.For example, this domain contains 24 amino acids in the VDR and 603amino acids in the MR. This domain is involved in transcriptionalactivation and in some cases its uniqueness may dictate selectivereceptor-DNA binding and activation of target genes by specific receptorisoforms. This domain can display synergistic and antagonisticinteractions with the domains of the LBD. For example, studies withmutated and/or deleted receptors show positive cooperativity of theamino and carboxy terminal domains. In some cases, deletion of either ofthese domains will abolish the receptor's transcriptional activationfunctions.

DNA-Binding Domain

The DBD is the most conserved structure in the nuclear receptorsuperfamily. It usually contains about 70 amino acids that fold into twozinc finger motifs, wherein a zinc ion coordinates four cysteines. DBDscontain two perpendicularly oriented α-helixes that extend from the baseof the first and second zinc fingers. The two zinc fingers function inconcert along with non-zinc finger residues to direct nuclear receptorsto specific target sites on DNA and to align receptor homodimer orheterodimer interfaces. Various amino acids in DBD influence spacingbetween two half-sites (usually comprised of six nucleotides) forreceptor dimer binding. For example, GR subfamily and ER homodimers bindto half-sites spaced by three nucleotides and oriented as palindromes.The optimal spacings facilitate cooperative interactions between DBDs,and D box residues are part of the dimerization interface. Other regionsof the DBD facilitate DNA-protein and protein-protein interactionsrequired for RXR homodimerization and heterodimerization on directrepeat elements.

The LBD may influence the DNA binding of the DBD, and the influence canalso be regulated by ligand binding. For example, TR ligand bindinginfluences the degree to which a TR binds to DNA as a monomer or dimer.Such dimerization also depends on the spacing and orientation of the DNAhalf sites. The receptors also can interact with other proteins andfunction to regulate gene expression.

The nuclear receptor superfamily has been subdivided into twosubfamilies: 1) GR (GR, AR, MR and PR) and 2) TR (TR, VDR, RAR, RXR, andmost orphan receptors) on the basis of DBD structures, interactions withheat shock proteins (hsp), and ability to form heterodimers. GR subgroupmembers are tightly bound by hsp in the absence of ligand, dimerizefollowing ligand binding and dissociation of hsp, and show homology inthe DNA half sites to which they bind. These half sites also tend to bearranged as palindromes. TR subgroup members tend to be bound to DNA orother chromatin molecules when unliganded, can bind to DNA as monomersand dimers, but tend to form heterodimers, and bind DNA elements with avariety of orientations and spacings of the half sites, and also showhomology with respect to the nucleotide sequences of the half sites. Bythis classification, ER does not belong to either-subfamily, since itresembles the GR subfamily in hsp interactions, and the TR subfamily innuclear localization and DNA-binding properties.

Ligand Binding Domain

The LBD is the second most highly conserved domain in these receptors.Whereas integrity of several different LBD sub-domains is important forligand binding, truncated molecules containing only the LBD retainnormal ligand-binding activity. This domain also participates in otherfunctions, including dimerization, nuclear translocation andtranscriptional activation, as described herein. Importantly, thisdomain binds the ligand and undergoes ligand-induced conformationalchanges as detailed herein.

Most members of the superfamily, including orphan receptors, possess atleast two transcription activation subdomains, one of which isconstitutive and resides in the amino terminal domain (AF-1), and theother of which (AF-2 (also referenced as TAU 4)) resides in theligand-binding domain whose activity is regulated by binding of anagonist ligand. The function of AF-2 requires an activation domain (alsocalled transactivation domain) that is highly conserved among thereceptor superfamily (approximately amino acids 1005 to 1022). Most LBDscontain an activation domain. Some mutations in this domain abolish AF-2function, but leave ligand binding and other functions unaffected.Ligand binding allows the activation domain to serve as an interactionsite for essential co-activator proteins that function to stimulate (orin some cases, inhibit) transcription.

For example, Shibata, H., et al. (Recent Progress in Hormone Res.52:141-164 (1997)) has reviewed the role of co-activators andco-repressors in steroid/thyroid hormone receptor systems. Steroidreceptor co-activator-one (SRC-1) appears to be a general co-activatorfor all AF-2 domain containing receptors tested. SRC-1 enhancestransactivation of steroid hormone-dependent target genes. Otherputative co-activators have been reported, including the SRC-1 relatedproteins, TIF-2 and GRIP-1, and other putative unrelated co-activatorssuch as ARA-70, Trip 1, RIP-140, and TIF-1. In addition anotherco-activator CREB-binding protein (CBP) has been shown to enhancereceptor-dependent target gene transcription. CBP and SRC-1 interact andsynergistically enhance trancriptional activation by the ER and PR. Aternary complex of CBP, SRC-1, and liganded receptors-may form toincrease the rate of hormone-responsive gene transcription.Co-repressors, such as SMRT and N—CoR, for TR and RAR, have beenidentified that also contribute to the silencing function of unligandedTR. The unliganded TR and RAR have been shown to inhibit basal promoteractivity; this silencing of target gene transcription by unligandedreceptors is mediated by these co-repressors. The collective datasuggests that upon binding of agonist, the receptor changes itsconformation in the ligand-binding domain that enables recruitment ofco-activators, which allows the receptor to interact with the basaltranscriptional machinery more efficiently and to activatetranscription. In contrast, binding of antagonists induces a differentconformational change in the receptor. Although some antagonist-boundreceptors can dimerize and bind to their cognate DNA elements, they failto dislodge the associated co-repressors, which results in anonproductive interaction with the basal transcriptional machinery.Similarly, the TR and RAR associate with co-repressors in the absence ofligand, thereby resulting in a negative interaction with thetranscriptional machinery that silences target gene expression. In thecase of mixed agonist/antagonists, such as 4-hydroxytamoxifen,activation of gene transcription may depend on the relative ratio ofco-activators and co-repressors in the cell or cell-specific factorsthat determine the relative agonistic or antagonistic potential ofdifferent compounds. These co-activators and co-repressors appear to actas an accelerator and/or a brake that modulates transcriptionalregulation of hormone-responsive target gene expression.

The carboxy-terminal activation subdomain, as described herein is inclose three dimensional proximity in the LBD to the ligand, so as toallow for ligands bound to the LBD to coordinate (or interact) withamino acid(s) in the activation subdomain. As described herein, the LBDof a nuclear receptor can be expressed, crystallized, its threedimensional structure determined with a ligand bound (either usingcrystal data from the same receptor or a different receptor or acombination thereof), and computational methods used to design ligandsto its LBD, including ligands that contain an extension moiety thatcoordinates the activation domain of the nuclear receptor.

Once a computationally designed ligand (CDL) is synthesized as describedherein and known in the art, it can be tested using assays to establishits activity as an agonist, partial agonist or antagonist, and affinity,as described herein. After such testing, the CDLs can be further refinedby generating LBD crystals with a CDL bound to the LBD. The structure ofthe CDL can then be further refined using the chemical modificationmethods described herein for three dimensional models to improve theactivity or affinity of the CDL and make second generation CDLs withimproved properties, such as that of a super agonist or antagonistdescribed herein. Agonist and antagonist ligands also can be selectedthat modulate nuclear receptor responsive gene transcription throughaltering the interaction of co-activators and co-repressors with theircognate nuclear hormone receptor. For example, CDL agonists can beselected that block or dissociate the co-repressor from interaction withthe receptor, and/or which promote binding or association of theco-activator. CDL antagonists can be selected that block co-activatorinteraction and/or promote co-repressor interaction with the targetreceptor. Selection can be done in binding assays that screen for CDLshaving the desired agonist or antagonist properties. Suitable assays forsuch screening are described herein and in Shibata, H., et al. (RecentProg. Horm. Res. 52:141-164 (1997)); Tagami, T., et al. (Mol. Cell Biol.17(5):2642-2648 (1997)); Zhu, X G., et al. (J. Biol. Chem.272(14):9048-9054 (1997)); Lin, B. C., et al. (Mol. Cell Biol.17(10):6131-6138 (1997)); Kakizawa, T., et al. (J. Biol. Chem.272(38):23799-23804 (1997)); and Chang, K. H., et al. (Proc. Natl. Acad.Sci. USA 94(17):9040-9045 (1997)), which references are incorporatedherein in their entirety by reference.

Nuclear Receptor Isoforms

The present invention also is applicable to generating new syntheticligands to distinguish nuclear receptor isoforms. As described herein,CDLs can be generated that distinguish between binding isoforms, therebyallowing the generation of either tissue specific or function specificsynthetic ligands. For instance, GR subfamily members have usually onereceptor encoded by a single gene, although are exceptions. For example,there are two PR isoforms, A and B, translated from the same mRNA byalternate initiation from different AUG codons. There are two GR forms,one of which does not bind ligand. This method is especially applicableto the TR subfamily which usually has several receptors that are encodedby at least two (TR: α, β) or three (RAR, RXR, and PPAR: α, β, γ) genesor have alternate RNA splicing and such an example for TR is describedherein.

Nuclear Receptor Crystals

The invention provides for crystals made from nuclear receptor ligandbinding domains with the ligand bound to the receptor. As exemplified inthe Examples, TRs are crystallized with a ligand bound to it. Crystalsare made from purified nuclear receptor LBDs that are usually expressedby a cell culture, such as E. coli. Preferably, different crystals(co-crystals) for the same nuclear receptor are separately made usingdifferent ligands, such as a naturally occurring ligand and at least onebromo- or iodo-substituted synthetic ligand that acts as an analog orantagonist of the naturally occurring ligand. Such bromo- andiodo-substitutions act as heavy atom substitutions in nuclear receptorligands and crystals of nuclear receptor proteins. This method has theadvantage for phasing of the crystal in that it bypasses the need forobtaining traditional heavy metal derivatives. After the threedimensional structure is determined for the nuclear receptor LBD withits ligand bound, the three dimensional structure can be used incomputational methods to design a synthetic ligand for the nuclearreceptor and further activity structure relationships can be determinedthrough routine testing using the assays described herein and known inthe art.

Expression and Purification of Other Nuclear Receptor LBD Structures

High level expression of nuclear receptor LBDs can be obtained by thetechniques described herein as well as others described in theliterature. High level expression in E. coli of ligand binding domainsof TR and other nuclear receptors, including members of thesteroid/thyroid receptor superfamily, such as the receptors ER, AR, MR,PR, RAR, RXR and VDR can also be achieved. Yeast and other eukaryoticexpression systems can be used with nuclear receptors that bind heatshock proteins as these nuclear receptors are generally more difficultto express in bacteria, with the exception of ER, which can be expressedin bacteria. Representative nuclear receptors or their ligand bindingdomains have been cloned and sequenced: human RAR-α, human RAR-γ, humanRXR-α, human RXR-β, human PPAR-α, human PPAR-β, human PPAR-γ, human VDR,human ER (as described in Seielstad et al., Molecular Endocrinology, vol9:647-658 (1995), incorporated herein by reference), human GR, human PR,human MR, and human AR. The ligand binding domain of each of thesenuclear receptors has been identified and is shown in FIG. 3. Using theinformation in FIG. 3 in conjunction with the methods described hereinand known in the art, one of ordinary skill in the art could express andpurify LBDs of any of the nuclear receptors, including those illustratedin FIG. 3, bind it to an appropriate ligand, and crystallize the nuclearreceptor's LBD with a bound ligand.

FIG. 3 is an alignment of several members of the steroid/thyroid hormonereceptor superfamily that indicates the amino acids to be included in asuitable expression vector.

Extracts of expressing cells are a suitable source of receptor forpurification and preparation of crystals of the chosen receptor. Toobtain such expression, a vector is constructed in a manner similar tothat employed for expression of the rat TR alpha (Apriletti et al.Protein Expression and Purification, 6:363-370 (1995), hereinincorporated by reference). The nucleotides encoding the amino acidsencompassing the ligand binding domain of the receptor to be expressed,for example the estrogen receptor ligand binding domain (hER-LBD)(corresponding to R at position 725 to L at position 1025 as standardlyaligned as shown in the FIG. 3), are inserted into an expression vectorsuch as the one employed by Apriletti et al (1995). For the purposes ofobtaining material that will yield good crystals it is preferable toinclude at least the amino acids corresponding to human TR-β positions725 to 1025. Stretches of adjacent amino acid sequences may be includedif more structural information is desired. Thus, an expression vectorfor the human estrogen receptor can be made by inserting nucleotidesencoding amino acids from position 700 to the c-terminus at position1071. Such a vector gives high yield of receptor in E. coli that canbind hormone (Seielstad et al. Molecular Endocrinology 9:647-658(1995)). However, the c-terminal region beyond position 1025 is subjectto variable proteolysis and can advantageously be excluded from theconstruct, this technique of avoiding variable proteolysis can also beapplied to other nuclear receptors.

TR-α and TR-β as Examples of Nuclear Receptor LBD Structure and FunctionTR Expression, Purification and Crystallization

As an example of nuclear receptor structure of the ligand binding domainthe α- and β-isoforms of TR are crystallized from proteins expressedfrom expression constructs, preferably constructs that can be expressedin E. coli. Other expression systems, such as yeast or other eukaryoticexpression systems can be used. For the TR, the LBD can be expressedwithout any portion of the DBD or amino-terminal domain. Portions of theDBD or amino-terminus can be included if further structural informationwith amino acids adjacent the LBD is desired. Generally, for the TR theLBD used for crystals will be less than 300 amino acids in length.Preferably, the TR LBD will be at least 150 amino acids in length, morepreferably at least 200 amino acids in length, and most preferably atleast 250 amino acids in length. For example the LBD used forcrystallization can comprise amino acids spanning from Met 122 to Val410 of the rat TR-α, Glu 202 to Asp 461 of the human TR-β.

Typically TR LBDs are purified to homogeneity for crystallization.Purity of TR LBDs is measured with sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE), mass spectrometry (MS) and hydrophobichigh performance liquid chromatography (HPLC). The purified TR forcrystallization should be at least 97.5% pure or 97.5%, preferably atleast 99.0% pure or 99.0% pure, more preferably at least 99.5% pure or99.5% pure.

Initially purification of the unliganded receptor can be obtained byconventional techniques, such as hydrophobic interaction chromatography(HPLC), ion exchange chromatography (HPLC), and heparin affinitychromatography.

To achieve higher purification for improved crystals of nuclearreceptors, especially the TR subfamily and TR, it will be desirable toligand shift purify the nuclear receptor using a column that separatesthe receptor according to charge, such as an ion exchange or hydrophobicinteraction column, and then bind the eluted receptor with a ligand,especially an agonist. The ligand induces a change in the receptor'ssurface charge such that when re-chromatographed on the same column, thereceptor then elutes at the position of the liganded receptor areremoved by the original column run with the unliganded receptor. Usuallysaturating concentrations of ligand are used in the column and theprotein can be preincubated with the ligand prior to passing it over thecolumn. The structural studies detailed herein indicate the generalapplicability of this technique for obtaining super-pure nuclearreceptor LBDs for crystallization.

More recently developed methods involve engineering a “tag” such as withhistidine placed on the end of the protein, such as on the aminoterminus, and then using a nickle chelation column for purification,Janknecht R., Proc. Natl. Acad. Sci. USA, 88:8972-8976-(1991)incorporated by reference.

To determine the three dimensional structure of a TR LBD, or a LBD fromanother member of the nuclear receptor superfamily, it is desirable toco-crystalize the LBD with a corresponding LBD ligand. In the case of TRLBD, it is preferable to separately co-crystalize it with ligands suchas T3, IpBr and Dimit that differ in the heavy atoms which they contain.Other TR ligands such as those encompassed by Formula 1 described hereinand known in the prior art, can also be used for the generation ofco-crystals of TR LBD and TR ligands. Of the compounds encompassed byFormula I it is generally desirable to use at least one ligand that hasat least one bromo- or iodo-substitution at the R₃, R₅, R₃′ or R₅′position, preferably such compounds will be have at least two suchsubstitutions and more preferably at least 3 such substitutions. Asdescribed herein, such substitutions are advantageously used as heavyatoms to help solve the phase problem for the three dimensionalstructure of the TR LBD and can be used as a generalized method ofphasing using a halogen (e.g. I or Br) substituted ligand, especiallyfor nuclear receptors.

Typically purified LBD, such as TR LBD, is equilibrated at a saturatingconcentration of ligand at a temperature that preserves the integrity ofthe protein. Ligand equilibration can be established between 2 and 37°C., although the receptor tends to be more stable in the 2-20° C. range.

Preferably crystals are made with the hanging drop methods detailedherein. Regulated temperature control is desirable to improve crystalstability and quality. Temperatures between 4 and 25° C. are generallyused and it is often preferable to test crystallization over a range oftemperatures. In the case of TR it is preferable to use crystallizationtemperatures from 18 to 25° C., more preferably 20 to 23° C., and mostpreferably 22° C.

Complexes of the TR-α LBD with a variety of agonists, including T₃,IpBr₂, Dimit, and Triac, are prepared with by methods described herein.For example, cocrystals of the rTR-α LBD, with ligand prebound, areprepared by vapor diffusion at ambient temperature from 15%2-methyl-2,4-pentanediol (MPD). The crystals are radiation sensitive,and require freezing to measure complete diffraction data. On a rotatinganode X-ray source, the crystals diffract to ˜3 Å; synchrotron radiationextends the resolution limit significantly, to as high as 2.0 Å for T₃cocrystals. The composition of the thyroid hormone, combined with theability to prepare and cocrystallize the receptor complexed with avariety of analogs, permitted the unusual phasing strategy. This phasingstrategy can be applied to the ligands of the nuclear receptorsdescribed therein by generating I and Br substitutions of such ligands.In this strategy, cocrystals of the TR LBD containing four hormoneanalogs that differ at the 3,5, and 3′ positions (T3, IpBr₂, Dimit, andTriac) provided isomorphous derivatives. For this set of analogs, thehalogen substituents (2Br and 3I atoms) function as heavy atoms, whilethe Dimit cocrystal (3 alkyl groups) acts as the parent. The initial 2.5Å multiple isomorphous replacement/anomalous scattering/density modifiedelectron density map allowed the LBD to be traced from skeletons createdin the molecular graphics program O5 (Jones, T. A. et al., ACTA Cryst,47:110-119 (1991), incorporated by reference herein). A model of the LBDwas built in four fragments, Arg157-Gly184, Trp186-Gly197,Ser199-Pro205, and Val210-Phe405, and refined in XPLOR using positionalrefinement and simulated annealing protocols. Missing residues werebuilt with the aid of difference density. The final model was refined toR_(cryst)=21.8% and R_(free)=24.4% for data from 15.0 to 2.2 Å, seeTable 6. The human TR-β LBD model was resolved by molecular replacementof the TR-α LBD coordinates. The structure is based on E202 to D461 witha his-tag at the N-terminus. The final model was refined toR_(cryst)=25.3% and R_(free)=28.9% for data from 30.0 to 2.4 Å+, seeTable 7.

This phasing strategy can be applied to the ligands of the nuclearreceptors described herein by generating I and Br substitutions of suchligands.

Three Dimensional Structure of TR LBD

Architecture of TR LBD

As an example of the three dimensional structure of a nuclear receptor,the folding of the TR-α₁ LBD is shown in FIG. 4. The TR-α LBD consistsof a single structural domain packed in three layers, composed of twelveα-helices, H1-12, and four short β-strands, S1-4, forming a mixedβ-sheet. The buried hormone and three antiparallel α-helices, H5-6, H9,and H10, form the central layer of the domain, as shown in FIG. 4. H1,H2, H3 and S1 form one face of the LBD, with the opposite face formed byH7, H8, H11, and H12. The first 35 amino acids of the N-terminus(Met122-Gln156) are not visible in the electron density maps. The threedimensional structure of the heterodimeric RXR:TR DNA-binding domainsbound to DNA, amino acids Met 122-Gln151 of the TR DBD make extensivecontacts with the minor groove of the DNA8. The five disordered aminoacids (Arg152-Gln156), which reside between the last visible residue ofthe TR DBD and the first visible residue of the LBD likely represent theeffective “hinge” linking the LBD and the DBD in the intact receptor.

The predominantly helical composition and the layered arrangement ofsecondary structure is identical to that of the unliganded hRXRα,confirming the existence of a common nuclear receptor fold between twonuclear receptors.

The TR LBD is visible beginning at Arg157, and continues in an extendedcoil conformation to the start of H1. A turn of α-helix, H2, covers thehormone binding cavity, immediately followed by short β-strand, S1,which forms the edge of the mixed β-sheet, parallel to S4, the outermostof the three antiparallel strands. The chain is mostly irregular untilH3 begins, antiparallel to H1. H3 bends at Ile221 and Ile222, residueswhich contact the ligand. The chain turns almost 90° at the end of H3 toform an incomplete α-helix, H4. The first buried core helix, H5-6,follows, its axis altered by a kink near the ligand at Gly 253. Thehelix is composed of mostly hydrophobic sidechains interrupted by twostriking exceptions: Arg262 is solvent inaccessible and interacts withthe ligand carboxylate (1-substituent), and Glu256 meets Arg329 from H9and Arg375 from H11 in a polar invagination. H5-6 terminates in a shortβ-strand, S2, of the four strand mixed sheet. S3 and S4 are joinedthrough a left-handed turn, and further linked by a salt bridge betweenLys284 and Asp272. Following S4, H7 and H8 form an L, stabilized by asalt bridge between Lys268 and Asp277. The turn between H7 and H8 adoptsan unusual conformation, a result of interaction with ligand and itsglycine rich sequence. H9 is the second core helix. antiparallel to theneighboring H5-6. Again, two buried polar sidechains are found, Glu315and Gln320. Glu315 forms a buried salt bridge with His358 and Arg356.The oxygen of Gln320 forms a hydrogen bond with the buried sidechain ofHis 175. The chain then switches back again to form H10, alsoantiparallel to H9. H11 extends diagonally across the full length of themolecule. Immediately after H11, the chain forms a type II turn, atapproximately 90° to H1. The chain then turns again to form H 12, whichpacks loosely against H3 and H11 as part of the hormone or ligandbinding cavity. The final five amino acids at the C-terminus,Glu406-Val410, are disordered. The architecture of the TR-β LBD isidentical to that of the TR-α LBD, with two significant differences. Anadditional helix is present at the N-terminus (residues Glu202-Ile208),which is part of the DBD, and packs antiparallel to H10. Following thehelix is a two residue turn (Gly209-His210) continuing into an extendedcoil to he start of H1, as seen in the TR-α LBD. A further differenceoccurs in the irregular conformation adopted between H2 and H3. In theTR-α LBD, residue Gly197-Asp211 form a loop that packs against thereceptor, contacting helices H7, H8, H11, and the loop between H11 andH12. In the TR-β LBD, only the ends of the loop are ordered, with thestretch Ala253-Lys263 disordered. In addition to these residues, theresidues of the His-tag at the N-terminus, and the final residue at theC-terminus, Asp461, are disordered.

TR LBD's Ligand Binding Cavity as an Example of a Nuclear Receptor'sBuried Ligand Cavity

The three dimensional structure of the TR LBD leads to the startlingfinding that ligand binding cavity of the LBD is solvent inaccessiblewhen a T3 or its isostere is bound to the LBD. This surprising resultleads to a new model of nuclear receptor three dimensional structure andfunction, as further described herein, particularly in the sectionselucidating the computational methods of ligand design and theapplication of such methods to designing nuclear receptor syntheticligands that contain extended positions that prevent normal activationof the activation domain.

Dimit, the ligand bound to the receptor, is an isostere of T₃ and athyroid hormone agonist. Therefore the binding of Dimit should reflectthat of T₃, and the Dimit-bound receptor is expected to be the activeconformation of TR. The ligand is buried within the receptor, providingthe hydrophobic core for a subdomain of the protein, as shown in FIG. 5a and b. H5-6 and H9 comprise the hydrophobic core for the rest of thereceptor.

An extensive binding cavity is constructed from several structuralelements. The cavity is enclosed from above by H5-6 (Met 256-Arg266),from below by H7 and H8 and the intervening loop (Leu287-Ile299), andalong the sides by H2 (185-187), by the turn between S3 and S4(Leu276-Ser277), by H3 (Phe215-Arg228), by H11 (His381-Met388) and byH12 (Phe401-Phe405). The volume of the cavity defined by these elements,calculated by GRASP (Columbia University, USA) (600 Å3), is essentiallythe volume of the hormone (530 Å). The change in volume can be exploitedfor ligand design as described herein. The remaining volume is occupiedby water molecules surrounding the amino-propionic acid substituent.FIG. 6 depicts various contacts (or interactions) between TR's LBD andthe ligand.

The planes of the inner and outer (prime ring) rings of the ligand arerotated from planarity about 60° with respect to each other, adoptingthe 3′-distal conformation (in which the 3′ substituent of the outerring projects down and away from the inner ring). The amino-propionicacid and the outer phenolic ring assume the transoid conformation, eachon opposite sides of the inner ring. The torsion angle χ₁ for theamino-propionic acid is 300°.

The amino-propionic acid substituent is packed loosely in a polar pocketformed by side chains from H2, H4 and S3. The carboxylate group formsdirect hydrogen bonds with the guanidium group of Arg228 and the amino Nof Ser277. In addition, Arg262, Arg266 and Asn179 interact with thecarboxylate through water-mediated hydrogen bonds. The three arginineresidues create a significantly positive local electrostatic potential,which may stabilize the negative charge of the carboxylate. No hydrogenbond is formed by the amino nitrogen. The interactions of theamino-propionic acid substituent are consistent with the fact thatTriac, which lacks the amino nitrogen, has a binding affinity equal tothat of T₃, indicating that the amino nitrogen and longer aliphaticchain of T₃ do not contribute greatly to binding affinity.

The biphenyl ether, in contrast, is found buried within the hydrophobiccore. The inner ring packs in a hydrophobic pocket formed by H3, H5-6,and S3. Pockets for the 3- and 5-methyl substituents are not completelyfilled, as expected since the van der waals radius of methyl substituentfor Dimit is smaller than the iodine substituent provided by the thyroidhormone T₃. Such pockets are typically 25 to 100 cubic angstroms(although smaller pocket for substitutes are contemplated in the 40 to80 cubic angstrom range) and could be filled more tightly with betterfitting chemical substitutions, as described herein.

The outer ring packed tightly in a pocket formed by H3, H5-6, H7, H8,H11 and H12, and the loop between H7 and H8. The ether oxygen is foundin a hydrophobic environment defined by Phe218, Leu287, Leu276, andLeu292. The absence of a hydrogen bond to the ether oxygen is consistentwith its role in establishing the correct stereochemistry of the phenylrings, as suggested by potent binding of hormone analogs withstructurally similar linkages possessing reduced or negligible hydrogenbonding capability. The 3′-isopropyl substituent contacts Gly290 and291. The presence of glycine at this position in the pocket can explainthe observed relationship between activity and the size of3′-substituents. Activity is highest for 3′-isopropyl, and decreaseswith added bulk. The only hydrogen bond in the hydrophobic cavity isformed between the phenolic hydroxyl and His381 Nε2. The conformation ofHis381 is stabilized by packing contacts provided by Phe405, and Met256.

The presence of a 5′ substituent larger than hydrogen affects thebinding affinity for hormone. The more abundant thyroid hormone,3,5,3′,5′-tetraiodo-L-thyronine (T₄), contains an iodine at thisposition, and binds the receptor with 2% of the affinity of T₃. Thestructure suggests that discrimination against T₄ is accomplishedthrough the combination of steric conflict by Met256 and possibly theconstraints imposed by the geometry of the hydrogen bond from His381 tothe phenolic hydroxyl. The 5′ position is a preferred location forintroducing a chemical modification of C—H at the 5′ of T3 or and TRagonist, as described herein, that produces an extension from the primering and results in the creation of an antagonist or partial agonist.

Deletion and antibody competition studies suggest the involvement ofresidues Pro162 to Val202 in ligand binding. The region does notdirectly contact hormone in the bound structure, although H2 packsagainst residues forming the polar pocket that interacts with theamino-propionic acid group. One role for H2, then, is to stabilize theseresidues in the bound state, H2, with β-strands S3 and S4, might alsorepresent a prevalent entry point for ligand, since the amino-propionicacid of the ligand is oriented toward this region. Studies of receptorbinding to T₃ affinity matrices demonstrate that only a linkage to theamino propionic acid is tolerated, suggesting that steric hindrancepresent in other linkages prevent binding. Furthermore, thecrystallographic temperature factors suggest the coil and β-strandregion is most flexible part of the domain FIG. 7. Participation of thisregion, part of the hinge domain between the DBD and LBD, in bindinghormone may provide structural means for ligand binding to influence DNAbinding, since parts of the Hinge domain contact DNA.

TR LBD Transcriptional Activation Helix as an Example of a NuclearReceptor Activation Domain

In addition to the startling finding that the ligand binding cavity issolvent inaccessible when loaded with a ligand, the activation helix ofTR LBD presents a surface to the ligand cavity for interaction betweenat least one amino acid and the bound ligand. The C-terminal 17 aminoacids of the TR, referred to as the activation helix or AF-2 (an exampleof an LBD activation domain), are implicated in mediatinghormone-dependent transcriptional activation. Although, mutations of keyresidues within the domain decrease ligand-dependent activation it wasunclear until the present invention whether such mutations directlyaffected ligand coordination. Although some mutations of this domainhave been noted to reduce or abolish ligand binding, other mutations inmore distant sites of the LBD have a similar effect.

Activation domains among nuclear receptors display an analogous threedimensional relationship to the binding cavity, which is a region of theLBD that binds the molecular recognition domain of a ligand, i.e. theactivation domain presents a portion of itself to the binding cavity(but necessarily the molecular recognition domain of the ligand). Manynuclear receptors are expected to have such domains, including theretinoid receptors, RAR and RXR, the glucocorticoid receptor GR, and theestrogen receptor ER. Based upon the TR's sequence, the domain isproposed to adopt an amphipathic helical structure. β-sheet or mixedsecondary structures, could be present as activation domains in lessrelated nuclear receptors.

Within the activation domain, the highly conserved motif ΦΦXEΦΦ, where Φrepresents a hydrophobic residue, is proposed to mediate interactionsbetween the receptors and transcriptional coactivators. Several proteinshave been identified which bind the TR in a hormone-dependent fashion.One of these, Trip1, is related to a putative yeast coactivator Sug1,and also interacts with both the C-terminal activation domain and asubset of the basal transcriptional machinery, suggesting a role intransactivation by the TR. Other proteins, such as RIP140, SRC1, (Onate,S. A. et. al., Science 270:1354-1357 (1995)) and TF-1 (see alsoLedouarim, B., et. al., EMBO J. 14:2020-2033 (1995)), and GRIP-1 (Heery,E., et al., Nature 387:733-736 (1997)) also interact with other nuclearreceptors in a ligand dependent manner through the C-terminal domain.Binding of these proteins can be modulated using the TR ligandsdescribed herein especially those TR ligands with extensions thatsterically hinder the interaction between the highly conserved motif andother proteins.

The C-terminal activation domain of the TR forms an amphipathic helix,H12, which nestles loosely against the receptor to form part of thehormone binding cavity. The helix packs with the hydrophobic residuesfacing inward towards the hormone binding cavity, and the chargedresidues, including the highly-conserved glutamate, extending into thesolvent, as shown in FIG. 8. The activation helix of TR LBD presents Phe401 to the ligand binding cavity and permits direct coordination withthe hormone i.e. such amino acids interact with the ligand forming a vander waals contact with the plane of the outer phenyl ring. Phe 405 alsointeracts with His 381, perhaps stabilizing its hydrogen bondingconformation, i.e. a favorable hydrogen bond interaction. Participationof Phe 401 and Phe 405 in binding hormone explains how mutation of theseresidues decreases hormone binding affinity. Furthermore, the impact ofthese mutations on activation likely derives from a role in stabilizingthe domain in the bound structure through increased hydrogen bondinteraction of dipole interactions. Glu 403 extends into the solvent,emphasizing its critical role in transactivation. In its observedconformation, presented on the surface as an ordered residue, against abackground of predominantly hydrophobic surface, Glu 403 is available tointeract with activator proteins described herein, as shown in FIG. 9.The other charged residues, Glu 405 and Asp 406 are disordered, as thehelix frays at Phe 405.

Two other sequences in the TR, τ2 and τ3, activate transcription whenexpressed as fusion proteins with a DNA-binding domain. The sequences,discovered in the TRB, correspond to TR-α residues Pro158-Ile168 in H1(τ2), and Gly290-Leu319 in H8 and H9 (τ3). Unlike the C-terminalactivation domain, τ2 and τ3 do not appear to represent modularstructural units in the rat TR-α LBD, nor present a surface forprotein-protein interactions: the critical aspartate/glutamate residuesof τ3 are located on two separate helices, and do not form a singlesurface; the charged residues of τ2 are engaged in ion pair interactionswith residues of the LBD. Thus, τ2 and τ3 may not function as activationdomains in the context of the entire receptor.

Computational Methods for Designing a Nuclear Receptor LBD LIGAND

The elucidation of the three dimensional structure of a nuclear receptorligand binding domain provides an important and useful approach fordesigning ligands to nuclear receptors using the computational methodsdescribed herein. By inspecting the FIGURES it can be determined thatthe nuclear receptor ligand is bound in a water inaccessible bindingcavity in the LBD and that chemical moieties can be added to selectedpositions on the ligand. Such chemical modifications, usuallyextensions, can fill up the binding cavity represented in the FIGURESfor a tighter fit (or less water) or can be used to disrupt or makecontacts with amino acids not in contact with the ligand before thechemical modification was introduced or represented in a figure of thethree dimensional model of the LBD. Ligands that interact with nuclearsuperfamily members can act as agonists, antagonists and partialagonists based on what ligand-induced conformational changes take place.

Agonists induce changes in receptors that place them in an activeconformation that allows them to influence transcription, eitherpositively or negatively. There may be several different ligand-inducedchanges in the receptor's conformation.

Antagonists, bind to receptors, but fail to induce conformationalchanges that alter the receptor's transcriptional regulatory propertiesor physiologically relevant conformations. Binding of an antagonist canalso block the binding and therefore the actions of an agonist.

Partial agonists bind to receptors and induce only part of the changesin the receptors that are induced by agonists. The differences can bequalitative or quantitative. Thus, a partial agonist may induce some ofthe conformation changes induced by agonists, but not others, or it mayonly induce certain changes to a limited extent.

Ligand-Induced Conformational Changes

As described herein, the unliganded receptor is in a configuration thatis either inactive, has some activity or has repressor activity. Bindingof agonist ligands induces conformational changes in the receptor suchthat the receptor becomes more active, either to stimulate or repressthe expression of genes. The receptors may also have non-genomicactions. Some of the known types of changes and/or the sequelae of theseare listed herein.

Heat Shock Protein Binding

For many of the nuclear receptors ligand binding induces a dissociationof heat shock proteins such that the receptors can form dimers in mostcases, after which the receptors bind to DNA and regulate transcription.

Nuclear receptors usually have heat shock protein binding domains thatpresent a region for binding to the LBD and can be modulated by thebinding of a ligand to the LBD. Consequently, an extended chemicalmoiety (or more) from the ligand that stabilizes the binding or contactof the heat shock protein binding domain with the LBD can be designedusing the computational methods described herein to produce a partialagonist or antagonist. Typically such extended chemical moieties willextend past and away from the molecular recognition domain on the ligandand usually past the buried binding cavity of the ligand.

Dimerization and Heterodimerization

With the receptors that are associated with the hsp in the absence ofthe ligand, dissociation of the hsp results in dimerization of thereceptors. Dimerization is due to receptor domains in both the DBD andthe LBD. Although the main stimulus for dimerization is dissociation ofthe hsp, the ligand-induced conformational changes in the receptors mayhave an additional facilitative influence. With the receptors that arenot associated with hsp in the absence of the ligand, particularly withthe TR, ligand binding can affect the pattern ofdimerization/heterodimerization. The influence depends on the DNAbinding site context, and may also depend on the promoter context withrespect to other proteins that may interact with the receptors. A commonpattern is to discourage monomer formation, with a resulting preferencefor heterodimer formation over dimer formation on DNA.

Nuclear receptor LBDs usually have dimerization domains that present aregion for binding to another nuclear receptor and can be modulated bythe binding of a ligand to the LBD. Consequently, an extended chemicalmoiety (or more) from the ligand that disrupts the binding or contact ofthe dimerization domain can be designed using the computational methodsdescribed herein to produce a partial agonist or antagonist. Typicallysuch extended chemical moieties will extend past and away from themolecular recognition domain on the ligand and usually past the buriedbinding cavity of the ligand.

DNA Binding

In nuclear receptors that bind to hsp, the ligand-induced dissociationof hsp with consequent dimer formation allows, and therefore, promotesDNA binding. With receptors that are not associated (as in the absenceof ligand), ligand binding tends to stimulate DNA binding ofheterodimers and dimers, and to discourage monomer binding to DNA.However, ligand binding to TR, for example, tends to decrease dimerbinding on certain DNA elements and has minimal to no effect onincreasing heterodimer binding. With DNA containing only a single halfsite, the ligand tends to stimulate the receptor's binding to DNA. Theeffects are modest and depend on the nature of the DNA site and probablyon the presence of other proteins that may interact with the receptors.Nuclear receptors usually have DBDs that present a region for binding toDNA and this binding can be modulated by the binding of a ligand to theLBD. Consequently, an extended chemical moiety (or more) from the ligandthat disrupts the binding or contact of the DBD can be designed usingthe computational methods described herein to produce a partial agonistor antagonist. Typically such extended chemical moieties will extendpast and away from the molecular recognition domain on the ligand andusually past the buried binding cavity of the ligand.

Repressor Binding

Receptors that are not associated with hsp in the absence of ligandfrequently act as transcriptional repressors in the absence of theligand. This appears to be due, in part, to transcriptional repressorproteins that bind to the LBD of the receptors. Agonist binding inducesa dissociation of these proteins from the receptors. This relieves theinhibition of transcription and allows the transcriptionaltransactivation functions of the receptors to become manifest.

Transcriptional Transactivation Functions

Ligand binding induces transcriptional activation functions in two basicways. The first is through dissociation of the hsp from receptors. Thisdissociation, with consequent dimerization of the receptors and theirbinding to DNA or other proteins in the nuclear chromatin allowstranscriptional regulatory properties of the receptors to be manifest.This may be especially true of such functions on the amino terminus ofthe receptors.

The second way is to alter the receptor to interact with other proteinsinvolved in transcription. These could be proteins that interactdirectly or indirectly with elements of the proximal promoter orproteins of the proximal promoter. Alternatively, the interactions couldbe through other transcription factors that themselves interact directlyor indirectly with proteins of the proximal promoter. Several differentproteins have been described that bind to the receptors in aligand-dependent manner. In addition, it is possible that in some cases,the ligand-induced conformational changes do not affect the binding ofother proteins to the receptor, but do affect their abilities toregulate transcription.

Nuclear receptors or nuclear receptor LBDs usually have activationdomains modulated in part by a co-activator/co-repressor system thatcoordinately functions to present a region for binding to DNA, and canbe modulated by the binding of a ligand to the LBD. Consequently, anextended chemical moiety (or more) from the ligand that disrupts thebinding or contact of the activation domain with co-activator and/orco-repressor can be designed using the computational methods describedherein to produce a partial agonist or antagonist. For instance, anagonist can be designed and/or selected which (1) blocks binding and/ordissociates co-repressor, and/or (2) promotes binding and/or associationof a co-activator. An antagonist can be designed which (1) promotesbinding and/or association of co-repressor, and/or (2) promotes bindingand/or association of co-activator. Ratios of agonists and antagonistsmay be used to modulate transcription of the gene of interest. Selectioncan be accomplised in binding assays that screen for ligands having thedesired agonist or antagonist properties, including such ligands whichinduce confomrational changes as decribed below. Suitable assays forsuch screening are described herein and in Shibata, H., et al. (RecentProg. Horm. Res. 52:141-164 (1997)); Tagami, T., et al. (Mol. Cell Biol.17(5):2642-2648 (1997)); Zhu, X G., et al. (J. Biol. Chem.272(14):9048-9054 (1997)); Lin, B. C., et al. (Mol. Cell Biol.17(10):6131-6138 (1997)); Kakizawa, T., et al. (J. Biol. Chem.272(38):23799-23804 (1997)); and Chang, K. H., et al. (Proc. Natl. Acad.Sci. USA 94(17):9040-9045 (1997)). Typically such extended chemicalmoieties will extend past and away from the molecular recognition domainon the ligand and usually past the buried binding cavity of the ligandand in the direction of the activation domain, which is often a helix asseen in the three dimensional model shown in the FIGURES in twodimensions on paper or more conveniently on a computer screen.

Ligand-Induced Conformational Change

Plasma proteins bind hormones without undergoing a conformational changethrough a static binding pocket formed between monomers or domains. Forexample, the tetrameric thyroid-binding plasma protein transthyretinforms a solvent-accessible hormone-binding channel at the oligomerinterface. The structure of the protein is unchanged upon bindinghormone with respect to the appearance of a buried binding cavity with aligand bound.

However, the structural role for a ligand bound to a nuclear receptorLBD, like rat TR-α LBD, predicts that the receptor would differ in thebound and unbound states. In the absence of hormone, the receptor wouldpossess a cavity at its core, uncharacteristic of a globular protein. Aligand (e.g. hormone) completes the hydrophobic core of the activereceptor after it binds to the nuclear receptor. Ligand binding by thereceptor is a dynamic process, which regulates receptor function byinducing an altered conformation.

An exact description of the hormone-induced conformational changesrequires comparison of the structures of the liganded and the unligandedTR. The structure of the unliganded human RXRα may substitute as a modelfor the unliganded TR. The rat TR-α LBD and human RXRα LBDs adopt asimilar fold, and it is likely that the structural similarity extends tothe conformational changes after ligand binding.

There are three major differences between the two structures, whichindeed appear to be the result of ligand binding. First, the bound ratTR-α LBD structure is more compact, with the hormone tightly packedwithin the hydrophobic core of the receptor. By contrast, the unligandedhuman RXRα LBD contains several internal hydrophobic cavities. Thepresence of such cavities is unusual in folded proteins, and is likely areflection of the unliganded state of the receptor. Two of thesecavities were proposed as possible binding sites for 9-cis retinoicacid, though these multiple sites only partly overlap with the singleburied binding cavity observed in the liganded rat TR-α LBD.

The second difference involves H11 in the rat TR-α LBD, whichcontributes part of the hormone binding cavity. H11, continuous in therat TR-α LBD, is broken at Cys 432 in the RXR, forming a loop betweenH10 and H11 in the hRXRα. This residue corresponds to His381 in the TR,which provides a hydrogen bond to the outer ring hydroxyl of the ligand.Furthermore, the hormone binding cavity occupied by ligand in the ratTR-α LBD is interrupted in the hRXRα by the same loop, forming anisolated hydrophobic pocket in the RXR with H6 and H7. In the bound ratTR-α LBD, the corresponding helices H7 and H8 are contiguous with thebinding pocket, and enclose the hormone binding cavity from below.

The third difference between the two receptors is the position of theC-terminal activation domain. While the C-terminal activation domainforms α-helices in both receptors, the domain in the rat TR-α LBDfollows a proline-rich turn, and lies against the receptor to contributepart of the binding cavity. In contrast, the activation domain in theunliganded hRXRα, is part of a longer helix which projects into thesolvent.

These differences lead to a model for an alternate conformation of theTR LBD assumed in the absence of ligand. In the unliganded TR, thesubdomain of the receptor surrounding the hormone binding cavity isloosely packed, with the binding cavity occluded by a partlyunstructured H11 providing a partial core for the receptor.

Upon binding hormone, residues which form a coil in the unbound receptorengage the ligand, and continues H11. The ordering of H11 could unblockthe hydrophobic cavity, allowing H7 and H8 to interact with hormone. Theextended hydrophobic cavity then collapses around the hormone,generating the compact bound structure.

It is possible to predict ligand-induced conformational changes in theC-terminal activation domain that rely, in part, on an extendedstructure in the unliganded TR that repacks upon ligand binding. Theligand-induced conformation change can be subtle since the amino acidsequence of the rat TR-α in the turn (393-PTELFPP-399) significantlyreduces the propensity of the peptide chain of the rat TR-α to form anα-helix and therefore repacking can be accomplished with a minor changein volume.

After the ligand-induced conformational change occurs, it is likely thatthe conformation of the C-terminal activation domain in the boundstructure changes packing compared to the unbound form of the receptor.Binding of the ligand improves the stability of the activation domain.The activation domain packs loosely even in the bound structure, asmeasured by the distribution of packing interactions for the entire LBD.The packing density for the activation domain, defined as the number ofatoms within 4.5 Å, is 1.5 standard deviations below the mean. Forcomparison, another surface helix, H1, is 0.5 standard deviations belowthe mean and the most poorly packed part of the structure, the irregularcoil from residues Ile196-Asp206, is 2.0 standard deviations below themean. Moreover, the majority of packing contacts for the C-terminaldomain in the bound receptor are provided either by residues whichinteract with ligand, such as His381, or by the ligand itself. Theconformation of these residues can be expected to be different in thebound and unbound receptors, and by extension the conformation ofC-terminal activation domain which relies upon these interactions.Without the stabilization provided by a bound ligand, it is likely thatthe C-terminal activation domain is disordered prior to hormone binding.

The interrelation of ligand-induced conformational changes is evident asdescribed herein. For example, His381 from H11 and Phe405 from H12interact in the bound structure to provide a specific hydrogen bond tothe phenolic hydroxyl. The ligand-induced changes which affect H11 andH12 are reinforcing, and lead to the formation of the compact, boundstate.

Comparison of the TR-α and TR-β LBD structures shows similar packing ofthe helices when complexed with the ligand Triac.

Computational Methods Using Three Dimensional Models and Extensions ofLigands

The three-dimensional structure of the liganded TR receptor isunprecedented, and will greatly aid in the development of new nuclearreceptor synthetic ligands, such as thyroid receptor antagonists andimproved agonists, especially those that bind selectively to one of thetwo TR isoforms (α or β). In addition, this receptor superfamily isoverall well suited to modern methods including three-dimensionalstructure elucidation and combinatorial chemistry such as thosedisclosed in EP 335 628, U.S. Pat. No. 5,463,564, which are incorporatedherein by reference. Structure determination using X-ray crystallographyis possible because of the solubility properties of the receptors.Computer programs that use crystallography data when practicing thepresent invention will enable the rational design of ligand to thesereceptors. Programs such as RASMOL can be used with the atomiccoordinates from crystals generated by practicing the invention or usedto practice the invention by generating three dimensional models and/ordetermining the structures involved in ligand binding. Computer programssuch as INSIGHT and GRASP allow for further manipulation and the abilityto introduce new structures. In addition, high throughput binding andbioactivity assays can be devised using purified recombinant protein andmodern reporter gene transcription assays described herein and known inthe art in order to refine the activity of a CDL.

Generally the computational method of designing a nuclear receptorsynthetic ligand comprises two steps:

1) determining which amino acid or amino acids of a nuclear receptor LBDinteracts with a first chemical moiety (at least one) of the ligandusing a three dimensional model of a crystallized protein comprising anuclear receptor LBD with a bound ligand, and

2) selecting a chemical modification (at least one) of the firstchemical moiety to produce a second chemical moiety with a structure toeither decrease or increase an interaction between the interacting aminoacid and the second chemical moiety compared to the interaction betweenthe interacting amino acid and the first chemical moiety.

As shown herein, interacting amino acids form contacts with the ligandand the center of the atoms of the interacting amino acids are usually 2to 4 angstroms away from the center of the atoms of the ligand.Generally these distances are determined by computer as discussed hereinand in McRee 1993, however distances can be determined manually once thethree dimensional model is made. Examples of interacting amino acids aredescribed in Appendix 2. See also Wagner et al., Nature378(6558):670-697 (1995) for stereochemical figures of three dimensionalmodels. More commonly, the atoms of the ligand and the atoms ofinteracting amino acids are 3 to 4 angstroms apart. The invention can bepracticed by repeating steps 1 and 2 to refine the fit of the ligand tothe LBD and to determine a better ligand, such as an agonist. As shownin the FIGURES the three dimensional model of TR can be represented intwo dimensions to determine which amino acids contact the ligand and toselect a position on the ligand for chemical modification and changingthe interaction with a particular amino acid compared to that beforechemical modification. Structural comparison of LBD isoforms complexedwith the same or similar ligand permit identification of fiducial andadjustable amino acids that can be exploited in designingisoform-specific ligands through chemical modification. “Fiducial”refers to amino acids that form rigid features of the ligand bindingcavity. “Adjustable” refers to amino acids that form less rigid featuresof the ligand binding cavity. The chemical modification may be madeusing a computer, manually using a two dimensional representation of thethree dimensional model or by chemically synthesizing the ligand. Thethree dimensional model may be made using Appendix 2 and the FIGURES. Asan additional step, the three dimensional model may be made using atomiccoordinates of nuclear receptor LBDs from crystallized protein as knownin the art, see McRee 1993 referenced herein.

The ligand can also interact with distant amino acids after chemicalmodification of the ligand to create a new ligand. Distant amino acidsare generally not in contact with the ligand before chemicalmodification. A chemical modification can change the structure of theligand to make as new ligand that interacts with a distant amino acidusually at least 4.5 angstroms away from the ligand. Often distant aminoacids will not line the surface of the binding cavity for the ligand, asthey are too far away from the ligand to be part of a pocket or surfaceof the binding cavity.

The interaction between an atom of a LBD amino acid and an atom of anLBD ligand can be made by any force or attraction described in nature.Usually the interaction between the atom of the amino acid and theligand will be the result of a hydrogen bonding interaction, chargeinteraction, hydrophobic interaction, van der waals interaction ordipole interaction. In the case of the hydrophobic interaction it isrecognized that this is not a per se interaction between the amino acidand ligand, but rather the usual result, in part, of the repulsion ofwater or other hydrophilic group from a hydrophobic surface. Reductionor enhancment of the interaction of the LBD and a ligand can be measuredby standard binding procedures, calculating or testing binding energies,computationally or using thermodynamic or kinetic methods as known inthe art.

Chemical modifications will often enhance or reduce interactions of anatom of a LBD amino acid and an atom of an LBD ligand. Steric hinderancewill be a common means of changing the interaction of the LBD bindingcavity with the activation domain. Chemical modifications are preferablyintroduced at C—H, C— and C—OH position in ligands, where the carbon ispart of the ligand structure which remains the same after modificationis complete. In the case of C—H, C could have 1, 2 or 3 hydrogens, butusually only one hydrogen will be replaced. The H or OH are removedafter modification is complete and replaced with the desired chemicalmoiety.

Because the thyroid receptor is a member of the larger superfamily ofhormone-binding nuclear receptors, the rules for agonist and antagonistdevelopment will be recognized by one skilled in the art as useful indesigning ligands to the entire superfamily. Examining the structures ofknown agonists and antagonists of the estrogen and androgen receptorssupports the generality of antagonist mechanism of action as shown inFIG. 10.

The overall folding of the receptor based on a comparison of thereported structure of the unliganded RXR and with amino acid sequencesof other superfamily members reveals that the overall folding ofreceptors of the superfamily is similar. Thus, it is predicted from thestructure that there is a general pattern of folding of the nuclearreceptor around the agonist or antagonist ligand.

The three dimensional structure of a nuclear receptor with a ligandbound leads to the nonobvious observation that a nuclear receptor foldsaround agonist ligands, as the binding cavity fits the agonist,especially the agonist's molecular recognition domain, and antagonistscommonly have chemical structures that extend beyond the ligand,especially the agonist, and would prohibit folding of the receptoraround the ligand to form a buried binding cavity or other groups thathave the same effect. The location of the extension could affect thefolding in various ways as indicated by the structure. Such extensionson antagonists are shown in FIG. 10 for various receptors and comparedto the corresponding agonist.

For example, an extension towards the carboxy-terminal activation helixaffects the packing/folding of this helix into the body of the receptor.This in turn can affect the ability of this portion of the nuclearreceptor to interact with other proteins or other portions of thereceptor, including transcriptional transactivation functions on theopposite end of the linear receptor, or the receptor's amino terminusthat may interact directly or indirectly with the carboxy-terminaltransactivation domain (including helix 12). Extensions in thisdirection can also affect the packing of helix 11 of TR (or itsanalogous helix in nuclear receptors) into the body of the receptor andselectively affect dimerization and heterodimerization of receptors. Anextension pointing towards helix 1 can affect the relationship of theDNA binding domain and hinge regions of the receptors with the ligandbinding domain and selectively or in addition affect the receptors'binding to DNA and/or interactions of receptors with proteins thatinteract with this region of the receptor. Other extensions towardshelix 11 can be made to affect the packing of this helix and helices 1and 10 and thereby homo- and hetero-dimerization. Such chemicalmodifications can be assessed using the computational methods describedherein. It is also possible that, in some cases, extensions may protrudethrough the receptor that is otherwise completely or incompletely foldedaround the ligand. Such protruding extensions could present a stericblockade to interactions with co-activators or other proteins.

The three dimensional structure with the ligand buried in the bindingcavity immediately offers a simple description of a nuclear receptorthat has a binding cavity that contains hinges and a lid, composed ofone or more structural elements, that move to accommodate and surroundthe ligand. The ligand to TR can be modified on specific sites withspecific classes of chemical groups that will serve to leave the lid andhinge region in open, partially open or closed states to achieve partialagonist or antagonist functions. In these states, the biologicalresponse of the TR is different and so the structure can be used todesign particular compounds with desired effects.

Knowledge of the three-dimensional structure of the TR-T3 complex leadsto a general model for agonist and antagonist design. An important novelfeature of the structural data is the fact that the T₃ ligand iscompletely buried within the central hydrophobic core of the protein.Other ligand-receptor complexes belonging to the nuclear receptorsuperfamily will have a similarly buried ligand binding site andtherefore this model will be useful for agonist/antagonist design forthe entire superfamily.

When design of an antagonist is desired, one needs either to preservethe important binding contacts of natural hormone agonist whileincorporating an “extension group” that interferes with the normaloperation of the ligand-receptor complex or to generate the requisitebinding affinity through the interactions of the extensions withreceptor domains.

The model applied to antagonist design and described herein is calledthe “Extension Model.” Antagonist compounds for nuclear receptors shouldcontain the same or similar groups that facilitate high-affinity bindingto the receptor, and in addition, such compounds should contain a sidechain which may be large and/or polar. This side chain could be anactual extension, giving it bulk, or it could be a side group with acharge function that differs from the agonist ligand. For example,substitution of a CH₃ for CH₂OH at the 21-position, and alteration atthe 11-position from an OH group to a keto group of cortisol generatesglucocorticoid antagonist activity (Robsseau, G. G., et. al., J. Mol.Biol. 67:99-115 (1972)). However, in most cases effective antagonistshave more bulky extensions. Thus, the antiglucocorticoid (andantiprogestin) RU486 contains a bulky side group at the 11-position(Horwitz, K. B. Endocrine Rev. 13:146-163 (1992)). The antagonistcompound will then bind within the buried ligand binding site of thereceptor with reasonably high affinity (100 nM), but the extensionfunction will prevent the receptor-ligand complex from adopting thenecessary conformation needed for transcription factor function. Theantagonism (which could be in an agonist or antagonist) may manifestitself at the molecular level in a number of ways, including bypreventing receptor homo/heterodimer formation at the HRE, by preventingcoactivator binding to receptor monomers, homodimers orhomo/heterodimers, or by a combination of these effects which otherwiseprevent transcription of hormone responsive genes mediated byligand-induced effects on the HRE. There are several antagonistcompounds for nuclear receptors in the prior art (see also Horwitz, K.B., Endocrine Rev. 13:146-163 (1992), Raunnaud J. P. et. al., J. SteroidBiochem. 25:811-833 (1986), Keiel S., et. al., Mol. Cell. Biol.14:287-298 (1994) whose antagonist function can be explained by theextension hypothesis. These compounds are shown in FIG. 10 along withtheir agonist counterparts. Each of these antagonists contains a largeextension group attached to an agonist or agonist analogue corestructure. Importantly, these antagonist compounds were discovered bychance and not designed with a structure-function hypothesis such as theextension principle.

One method of design of a thyroid antagonist using the extensionhypothesis is provided below as a teaching example. Thethree-dimensional structure of the TR-α Dimit complex combined withstructure-activity data published in the prior art, especially thosereference herein, can be used to establish the following ligand-receptorinteractions which are most critical for high-affinity ligand binding. Aphysical picture of these interactions is shown in FIG. 6. The figuredescribes the isolated essential contacts for ligand binding. Becausethe ligand is buried in the center of the receptor, the structuralspacing between these isolated interactions is also important. Thus, ourpresent knowledge of this system dictates that, for this example, anewly designed ligand for the receptor must contain a thyroninestructural skeleton, or two substituted aryl groups joined by a one-atomspacer.

The general structure for an antagonist designed by the extensionhypothesis is exemplified in the following general description of thesubstituents of a TR antagonist (referring to Formula 1): R1 can haveanionic groups such as a carboxylate, phosphonate, phosphate, sulfate orsulfite and is connected to the ring with a 0 to 3 atom linker,comprising one or more C, O, N, S atoms, and preferably a 2 carbonlinker. Such R1 can be optionally substituted with an amine (e.g. —NH2).R3 and R5 are small hydrophobic groups such as —Br, —I, or —CH3. R3 andR5 can be the same substituents or different. R₃′ can be a hydrophobicgroup that may be larger than those of R3 and R5, such as —I, —CH3,-isopropyl, -phenyl, -benzyl, 5 and 6 ring heterocycles. R₄′ is a groupthat can participate in a hydrogen bond as either a donor or acceptor.Such groups include —OH, —NH2, and —SH. R₅′ is an important extensiongroup that makes this compound an antagonist. R₅′ can be a long chainalkyl (e.g. 1 to 9 carbons, straight chain or branched), aryl (benzyl,phenyl and substituted benzyl and phenyl rings (e.g with halogen, alkyl(1 and 5 carbons) and optionally connected to the ring by a —CH2-),heterocycle (e.g. 5 or 6 atoms, preferably 5 carbons and 1 nitrogen, orfive carbons), which can optionally include polar (e.g. —OH, —NH₂, and—SH), cationic (e.g. —NH3, N(CH)3), or anionic (carboxylate,phosphonate, phosphate or sulfate) groups. R₅′ can also be a polar (e.g.—OH, —NH₂, and —SH), cationic (e.g. —NH3, —N(CH3)3), and anionic(carboxylate, phosphonate, phosphate or sulfate) groups. X is the spacergroup that appropriately positions the two aromatic rings. This group isusually a one-atom spacer, such as O, S, SO, SO2, NH, NZ where Z is analkyl, CH2, CHOH, CO, C(CH3)OH, and C(CH3)(CH3). X also may be NR₇, CH,CR₇, R₇, where R₇, is an alkyl, aryl or 5- or 6-membered heterocyclicaromatic. R2, R6, R2′ and R6′ can be —F, and —Cl and are preferably H.

A TR ligand can also be described as a substituted phenylated 3,5 diiodotyrosine with substituted R5′ and R3′ groups. R5′ can be a long chainalkyl (e.g. 4 to 9 carbons, straight chain or branched), aryl (benzyl,phenyl and substituted benzyl and phenyl rings (e.g with halogen, alkyl(1 and 5 carbons) and optionally connected to the ring by a —CH2-),heterocycle (e.g. 5 or 6 atoms, preferably 5 carbons and 1 nitrogen, orfive carbons), which can optionally include polar (e.g. —OH, —NH2, and—SH), cationic (e.g. —NH3, N(CH)3), or anionic (carboxylate,phosphonate, phosphate or sulfate) groups. R5′ can also be a polar (e.g.—OH, —NH₂, and —SH), cationic (e.g. —NH3, N(CH)3), and anionic(carboxylate, phosphonate, phosphate or sulfate) groups. R3′ can be-IsoPr, halogen, —CH3, alkyl (1 to 6 carbons) or aryl (benzyl, phenyland substituted benzyl and phenyl rings (e.g with halogen, alkyl (1 and5 carbons) and optionally connected to the ring by a —CH2-), heterocycle(e.g. 5 or 6 atoms, preferably 5 carbons and 1 nitrogen, or fivecarbons), which can optionally include polar (e.g. —OH, —NH2, and SH),cationic (e.g. —NH3, N(CH)3), or anionic (carboxylate, phosphonate,phosphate or sulfate) groups.

A TR antagonist can also be a modified T₃ agonist (having a biphenylstructure) wherein R₅′ is alkyl, aryl, 5- or 6-membered heterocyclicaromatic, heteroalkyl, heteroaryl, arylalkyl, heteroaryl alkyl,polyaromatic, polyheteroaromatic, polar or charged groups, wherein saidR₅′ may be substituted with polar or charged groups. The R5′ groups aredefined, as described herein.

Using these methods the ligands of this example preferably have thefollowing properties:

-   -   1. The compounds should bind to the TR with high affinity (for        example 100 nM).    -   2. The compounds should bind the receptor in the same basic        orientation as the natural hormone.    -   3. The extension group R5′ should project toward the activation        helix (C-terminal helix) of the receptor.    -   4. The appropriate substituent at R5′ should perturb the        activation helix from its optimal local structure needed for        mediating transcription.

Antagonists may also be designed with multiple extensions in order toblock more than one aspect of the folding at any time.

TR ligands (e.g. super agonists) can be designed (and synthesized) toenhance the interaction of at least one amino acid with at least onechemical moiety on the ligand's molecular recognition domain. One methodis to enhance the charge and polar interactions by replacing thecarboxylate of T₃ (R1 position) with phosphonate, phosphate, sulfate orsulfite. This enhances the interaction with Arg 262, Arg 266 and Arg228. The interaction of at least one amino acid with at least onechemical moiety on the ligand's molecular recognition domain can also beenhanced by increasing the size of R1 group to fill the space occupiedby water when Dimit is bound (referring to R1). Preferably the group hasa complementary charge and hydrophobicity to the binding cavity.

Another way of improving the interaction of at least one amino acid withat least one chemical moiety on the ligand's molecular recognitiondomain is to restrict the conformation of the dihedral angle between thetwo phenyl rings of the thyronine ligand in solution. In solution theplanes of two phenyl rings are orthogonal where the dihedral angle is90°. In the TR Dimit structure, the dihedral angle is close to 60°. A TRligand design that fixes the angle between the two phenyl rings willlead to tighter binding. Such a ligand may be made by connecting the R6′and the R5 positions of a thyronine or a substituted thyronine-likebiphenyl. The size of the cyclic connection can fix the angle betweenthe two phenyl rings. Referring specifically to Formula 1, the followingcyclic modifications are preferred: 1) R₅ is connected to R₆′, 2) R₃ isconnected to R₂′ or 3) R₅ is connected to R₆′ and R₃ is connected toR2′. The connections can be made by an alkyl or heteroalkyl chain havingbetween 1 to 6 atoms and preferably from 2 to 4 carbon atoms or otheratoms. Any position of the heteroalkyl chain can be N, O, P or S. The Sand P heteroatoms along said heteroalkyl chain are in any of theirpossible oxidative states. The N heteroatom or any carbon along thealkyl or heteroalkyl chain may have one or more Z substituents, whereinZ is alkyl, heteroalkyl, aryl, heteroaryl, 5- or 6-membered heterocyclicaromatic. These compounds can be claimed with the proviso that Formula 1does not include any prior art compound as of the priority filing dateof this application.

The interaction of at least one amino acid with at least one chemicalmoiety on the ligand's molecular recognition domain can also be enhancedby selecting a chemical modification that fills the unfilled spacebetween a TR ligand and the LBD in the area of the bridging oxygen (suchas in T3, Triac or Dimit). Thus, a slighter larger moiety that replacesthe ether oxygen can enhance binding. Such a linker may be a mono- orgeminal-disubstituted carbon group. A group approximately the same sizeas oxygen but with greater hydrophobicity is preferred as well as small,hydrophobic groups for the disubstituted carbon.

Compounds of Formula I or derivatives thereof that modulate TR activityalso may be designed and selected to interact with a conformationallyconstrained structural feature of a TR LBD that is conserved among TRLBD isoforms to increase TR-specific selectivity. Conserved structuralfeatures of a TR LBD include residues found in equivalent positions ofTR LBD isoforms which interact with a conserved structural feature of acompound comprising the biphenyl scaffold (φ-X-φ) or a single phenylscaffold (φ-X or X-φ) of Formula I. Conformationally constrainedstructural features of a TR LBD include residues that have their naturalflexible conformations fixed by various geometric and physical-chemicalconstraints, such as local backbone, local side chain, and topologicalconstraints. These types of constraints are exploited to restrictpositioning of atoms involved in receptor-ligand recognition andbinding. For example, comparison of atomic models of TR LBD isoformsbound to thyronine and thyronine-like ligands reveal that certainresidues which contact the ligands are restricted to particulartopological shapes and angles of rotation about bonds. These includeMet259, Leu276, Leu292, His381, Gly290, Ile221, and Phe401 of TR-α. Thecorresponding positions in TR-β include Met313, Leu330, Leu346, His435,Gly344, Ile275 and Phe455, respectively.

Selectivity imparted by conformationally constrained features of boththe receptor and compound are of particular interest. For example,compounds of Formula I comprising constrained cyclic carbons andsubstituent groups that interact with a constrained feature of a TR LBDcan be exploited to further increase binding specificity while reducingthe potential for cross-over interaction with other receptors. Theseinclude hydrophobic and/or hydrophilic contacts between constrainedresidues of a TR LBD and atomic groups of the following constituents ofthe compound in reference to Formula I: (i) the biphenyl rings; (ii) theR3-substituent; (iii) the R3′-substituent; and (iv) the R4′-substituent.

For example, contacts to the phenyl moiety comprising the R1, R2, R3, R5and R6 substituents, i.e., the ring proximal to the polar pocket (the“inner ring”), include a cycle carbon atom that interacts with an atomof a hydrophobic residue of a TR LBD, such as a carbon and oxygen atomof Met259 and a carbon atom of Leu276 of TR-α, or Met313 and Leu330 ofTR-β, where the cycle carbon is about 3.0 to 4.0 A from the atom of thehydrophobic group. For example, comparison of TR-α complexed with T3 andTR-β complexed with GC-1 reveals the following conserved inner ringcontacts: Ligand TR LBD T3/Atom TR-α Residue Atom Distance C11 Met259 C3.95 C11 Met259 O 3.59 C11 Met259 CB 3.77 C7 Leu276 CD2 3.80 C9 Leu276CD2 3.70 GC1/Atom TR-β Residue Atom Distance C11 Met313 C 3.85 C11Met313 O 3.41 C11 Met313 GB 3.79 C7 Leu330 CD2 3.56 C9 Leu330 CD2 3.63

Contacts to the phenyl moiety comprising the R2′, R3′, R4′, R5′ and R6′substituents, i.e., the ring distal to the polar pocket (the “outerring”), include a cyclic carbon atom that interacts with an atom of ahydrophobic residue of a TR LBD, such as a carbon atom of Leu292 ofTR-α, or Leu346 of TR-β, where the cyclic carbon atom is about 3.0 to4.0 A from the atom of the hydrophobic residue. For example, comparisonof TR-α complexed with T3 and TR-β complexed with GC-1 reveals thefollowing conserved outer ring contacts: Ligand TR LBD T3/Atom TR-αResidue Atom Distance C6 Leu292 CD2 3.58 C8 Leu292 CD2 3.50 GC1/AtomTR-β Residue Atom Distance C6 Leu346 CD2 3.77 C8 Leu346 CD2 3.80

Contacts to the R3-substituent include an atom that interacts with acarbon atom of a hydrophobic residue of a TR LBD, such as Ile221 ofTR-α, or Ile275 of TR-β, where the R3-substituent atom is about 3.0 to4.0 A from the carbon atom of the hydrophobic residue. For example,comparison of TR-α complexed with T3 and TR-β complexed with GC-1reveals the following conserved R3-substituent contacts: Ligand TR LBDT3/Atom TR-α Residue Atom Distance I1 Ile221 CG1 4.01 GC1/Atom TR-βResidue Atom Distance C19 Ile275 CG1 3.98

Contacts to the R3′-substituent include an atom that interacts with anatom of a hydrophobic or hydrophilic residue of a TR LBD, such as anoxygen atom of Gly290 of TR-α, or Gly344 of TR-β, where theR3′-substituent atom is about 3.0 to 4.0 A from the atom of thehydrophobic or hydrophilic residue. For example, comparison of TR-αcomplexed with T3 and TR-β complexed with GC-1 reveals the followingconserved R4′-substituent, phenolic hydroxyl contacts: Ligand TR LBDT3/Atom TR-α Residue Atom Distance I2 Gly290 O 3.50 GC1/Atom TR-βResidue Atom Distance C18 Gly344 O 3.60

Contacts to the R4′-substituent comprising a phenolic hydroxyl includecarbon and oxygen atoms that interact with a hydrophobic or hydrophilicresidue of a TR LBD, such as a carbon and nitrogen atom of His381 ofTR-α, or His435 of TR-β, where the R4′-substituent atom is about 2.0 to4.0 A from an atom of the hydrophobic or hydrophilic residue. Forexample, comparison of TR-α complexed with T3 and TR-β complexed withGC-1 reveals the following conserved R4′-substituent, phenolic hydroxylcontacts: Ligand TR LBD T3/Atom TR-α Residue Atom Distance C10 His381CD2 3.97 O1 His381 CD2 3.39 O1 His381 CE1 3.82 C8 His381 NE2 3.47 C10His381 NE2 3.55 O1 His381 NE2 2.70 GC1/Atom TR-β Residue Atom DistanceC10 His435 CD2 3.89 O1 His435 CD2 3.64 O1 His435 CE1 3.79 C8 His435 NE23.44 C10 His435 NE2 3.33 O1 His435 NE2 2.77

Contacts to the R4′-substituent also may include an atom that interactswith a carbon atom of a hydrophobic residue of a TR LBD, such as Phe401of TR-α, or Phe455 of TR-β, for defining agonist activity, i.e., properpresentation of helix-12 (H12) of the TR LBD following ligand binding.The R4′-substituent atom is about 3.0 to 4.0 A from the carbon atom ofthe hydrophobic group. For example, comparison of TR-α complexed with T3and TR-β complexed with GC-1 reveals the following conservedR4′-substituent contacts: Ligand TR LBD T3/Atom TR-α Residue AtomDistance O1 Phe401 CE1 3.52 O1 Phe401 CZ 3.32 GC1/Atom TR-β Residue AtomDistance O1 Phe455 CE1 3.40 O1 Phe455 CZ 3.22

Comparison of atomic models of TR LBD isoforms complexed with the sameand/or different ligands therefore facilitates the identification of newcompounds that fit spacially and preferentially into a TR LBD. Modeling,comparison of TR-ligand overlays, and comparison of TR LBD isoforms alsopermit identification of conformationally conserved structural featuresof TR LBD/ligand contacts. Exploiting conformational constraints of theLBD-ligand interaction identified by such methods therefore improves thedesign and identification of new compounds having increased selectivityfor binding a particular type of nuclear receptor, such as TR.

TR-α and TR-β Selectivity for the Thyroid Hormone Receptor

Using the method described herein ligands can be designed thatselectively bind to the alpha more than the beta TR or vice versa. TheX-ray crystallographic structure of the rat TR-α LBD provides insightinto design of such ligands.

The three dimensional structure reveals that the major differencebetween the TR-α and TR-β in the ligand binding cavity resides in aminoacid Ser 277 (with the side group —CH2OH) in the rat TR-α and whosecorresponding residue is 331, asparagine (with the side group—CH2CONH2), in the human TR-β. The side chain in human TR-β is larger,charged and has a different hydrogen bonding potential, which wouldallow the synthesis of compounds that discriminate between thisdifference. The Ser277 (Asn331 in TR-β) forms part of the polar pocketof the TR LBD, indicating that for TR-α versus TRAM-β discrimination,ligands can be designed to contain chemical modification of theR1-substitutent with reference to Formula I that exploit thisdifference.

For example, in the complex of TR-α with Triac, Ser277 does notparticipate in ligand binding. The absence of a role for Ser277 (Asn331in beta) is consistent with the equal affinity of Triac for the alphaand beta isoforms, and indirectly supports the contention thatalpha/beta selectivity resides in the amino acid substitution Ser277 toAsn331 and its interaction with Arg228. The effect of the amino acidsubstitution is further evident when the interactions of Asn331 andArg282 in the structures of the TR-β LBD complexed with GC-1 or Triacare compared with those of Ser277 and Arg228 in the TR-α LBD. In thecomplex with GC-1, Asn331 forms a hydrogen bond to Arg282, which in turnforms a hydrogen bond with the carboxylate of GC-1, a pattern thatresembles the interactions of Ser277 and Arg228 in the complexes of theTR-α LBD complexed with T₃ or Triac. However, in the complex of TR-βwith Triac, Arg282 rotates away from Asn331 and the ligand, insteadforming hydrogen bonds to residues Thr287 and Asp291 of H3. Therefore,differences exist between the two isoforms in the conformation of thepolar pocket, depending on the nature of the ligand R1-substitutent,indicating that certain substituents may interact preferentially withthe conformation of a given isoform.

Comparing overlays of various ligands bound to the TR-α versus TR-β LBDsshows the positioning of the ligand to be very similar. Surprisingly,comparison of the volume and area for the TR-α and TR-β LBDs bound bythe same or different ligands unexpectedly shows that the cubic space orvolume available for accommodating ligand binding by the TR-β LBD(645±28.28 Å³) is larger and more flexable than that of the TR-α LBD(596.25±7.97 Å³) (Table 1). The volume of the ligand binding cavity forTR-α varies over a narrow range of about 8+, with a maximum differenceof about 16+. In contrast, the volume of the ligand binding cavity forTR-β differs by nearly 40+ between the complexes with GC-1 and Triac.There also is a difference in the volume of the ligand binding cavitywhen comparing the same ligand bound to TR-α and TR-β. For example, TR-αand TR-β complexed with Triac differ in LBD volume by about 36 Å³.Comparison of TR-α and TR-β bound to Dimit and GC-1, respectively, whichligands have similar volume/area and superpositioned architecture, showthat the difference in LBD volume is about 75 Å³. These differences areattributed primarily to variable movement and interaction of side chaingroups with ligand substitutents of the phenyl moiety (φ) of thebiphenyl scaffold (φ-X-φ) located proximal to the polar pocket, e.g.,R1-substituents in reference to Formula I. In contrast, the volumeavailable in the hydrophobic pocket for both the TR-α and TR-β LBDs issubstantially the same. For example, binding of Triac to the TR-β LBDdisplaces the side chain of Arg 282 providing approximately 60 Å³ in thepolar pocket cavity, exposing the polar pocket to bulk solvent exchange.For GC1 bound to the TR-β LBD, approximately 14 Å³ is due to side chainmotion of Met310, and approximately 44 Å³ is due to side chain motion ofArg320, the combination of which increases the size of the polar pocketin the TR-β LBD. This extra pliability also may explain the absence ofordered water in the polar pocket of TR-β LBD bound to Triac or GC-1,which is in contrast to the ordered water found in the polar pocket ofTR-α LBD bound to Dimit, IpBr2 or T3. TABLE 1* rTR-α Dimit Triac IpBr2T3 TR LBD (volÅ³/areaÅ²) 590/456 589/440 601/474 605/472 Ligand(volÅ³/areaÅ²) 303/314 333/326 326/330 355/346 Complementarity 0.65 0.680.66 0.71 hTR-β GC-1 Triac TR LBD (volÅ³/areaÅ²) 665/575 625/474 Ligand(volÅ³/areaÅ²) 294/310 333/326 Complementarity 0.61 0.67*TR LBD volume and area are reported in Angstroms measured by GRASP.Complementarity is determined as defined in Lawrence et al., J. Mol.Biol. 234:946-950 (1993).

Residue Ser277 in TR-α and the corresponding residue Asn331 of TR-β alsocontribute to the volumetric differences observed in the polar pocketsof these two TR isoforms. And substitution of the Asn331 of hTR-β withserine has the affect of modifying ligand binding affinity of TR-β sothat it resembles that of TR-α (See Example 5). Taken together,differences in hydrogen bonding of atoms of the side chain group ofSer277 in TR-α and Asp331 in TR-β extending from the equivalent backboneposition in these TR LBDs and the more restricted polar pocket of theTR-α LBD further supports the concept of designing TR LBDisoform-specific ligands having substitutents that fit spacially andpreferentially into the polar pocket of either the TR-α or TR-β LBDs.Exploitation of this difference provides an additional means forcomputational design of isoform-specific TR agonists and antagonists.

In terms of ligand design, these differences mean that for β selectiveligands, some or all of the following differences should be exploited:

-   -   1. The presence of a larger side chain asparagine.    -   2. The ability of the carbonyl group on the side chain to        provide a strong hydrogen bond acceptor.    -   3. The ability of the amido group on the side chain to provide a        two hydrogen bond donors.    -   4. Adjustment of polarity to reorganize the trapped water in the        T3 pocket.    -   5. Greater size and flexibility of the polar pocket.

In terms of pharmaceutical design, these differences mean that forα-selective ligands, some or all of the following differences should beexploited:

-   -   1. The presence of a smaller side group.    -   2. The ability of the hydroxyl on the —CH₂OH side group carbonyl        group on the side chain to provide a weak hydrogen donor.    -   3. Adjustment of polarity to reorganize the trapped water in the        T3 pocket.    -   4. Smaller size and limited flexibility of the polar pocket.

In both cases these differences can be exploited in a number of ways.For example, they can also be used with a software set for constructionof novel organic molecules such as LUDI from Biosym-MSI. An example ofdesigning TR-β selective ligands is increasing the polarity of a ligandsubstituent located in the polar pocket of a TR LBD through addition ofone or more ligand groups having a formal negative charge and/ornegative dipole charge that interacts with a formal positive chargeand/or positive dipole charge of a group in the polar pocket of the LBD.This exploits preferential interactions, such as with the additionalpositive charge contributed by Asn 331 in TR-β. Another example of aTR-β selective ligand is one that comprises one or more groups which fitspacially into the TR-β LBD polar pocket. This exploits spacialdifferences between TR LBD isoforms, such as the larger and moreflexible polar pocket of TR-β.

Methods of Treatment

The compounds of Formula 1 can be useful in medical treatments andexhibit biological activity which can be demonstrated in the followingtests:

(i) the induction of mitochondrial α-glycerophosphate dehydrogenase(GPDH:EC 1.1.99.5). This assay is particularly useful since in certainspecies e.g. rats it is induced specifically by thyroid hormones andthyromimetics in a close-related manner in responsive tissues e.g.liver, kidney and the heart (Westerfield, W. W., Richert, D. A. andRuegamer, W. R., Endocrinology (1965) 77:802). The assay allows directmeasurement in rates of a thyroid hormone-like effect of compounds andin particular allows measurement of the direct thyroid hormone-likeeffect on the heart. Other measurements included parameters such asheart rate and cardiac enzymes including Ca⁺⁺ ATPase, Na⁺⁺/K⁺ ATPase,myosin isoforms and specific liver enzymes;

(ii) the elevation of basal metabolic rate as measured by the increasein whole body oxygen consumption (see e.g., Barker et al., Ann. N.Y.Acad. Sci., (1960) 86:545-562);

(iii) the stimulation of the rate of beating of atria isolated fromanimals previously dosed with thyromimetrics (see e.g., Stephan et al.,Biochem. Pharmacol. (1992) 13:1969-1974; Yokoyama et al., J. Med. Chem.,(1995) 38:695-707);

(iv) the change in total plasma cholesterol levels as determined using acholesterol oxidase kit (for example, the Merck CHOD iodine colorimetrickit. see also, Stephan et al. (1992));

(v) the measurement of LDL (low density lipoprotein) and HDL (highdensity lipoprotein) cholesterol in lipoprotein fractions separated byultracentrifigation; and p (vi) the change in total plasma triglyceridelevels as determined using enzymatic color tests, for example the MerckSystem GPO-PAP method.

The compounds of Formula 1 can be found to exhibit selectivethyromimetic activity in these tests,

(a) by increasing the metabolic rate of test animals, and raisinghepatic GPDH levels at doses which do not significantly modify cardiacGPDH levels.

(b) by lowering plasma cholesterol and triglyceride levels, and theratio of LDL to HDL cholesterol at doses which do not significantlymodify cardiac GPDH levels.

The compounds of Formula 1 may therefore be used in therapy, in thetreatment of conditions which can be alleviated by compounds whichselectively mimic the effects of thyroid hormones in certain tissueswhilst having little or no direct thyromimetic effect on the heart. Forexample, compounds of Formula 1 which raise hepatic GPDH levels andmetabolic rate at doses which do not significantly modify cardiac GPDHlevels are indicated in the treatment of obesity.

Agonists of Formula 1 will lower total plasma cholesterol, the ratio ofLDL-cholesterol to HDL-cholesterol and triglyceride levels at doseswhich do not significantly modify cardiac GPDH levels are indicated foruse as general antihyperlipidaemic (antihyperlipoproteinaemic) agentsi.e. in the treatment of patients having elevated plasma lipid(cholesterol and triglyceride) levels. In addition, in view of thiseffect on plasma cholesterol and triglyceride, they are also indicatedfor use as specific anti-hypercholesterolemic andanti-hypertriglyceridaemic agents.

Patients having elevated plasma lipid levels are considered at risk ofdeveloping coronary heart disease or other manifestations ofatherosclerosis as a result of their high plasma cholesterol and/ortriglyceride concentrations. Further, since LDL-cholesterol is believedto be the lipoprotein which induces atherosclerosis, and HDL-cholesterolbelieved to transport cholesterol from blood vessel walls to the liverand to prevent the build up of atherosclerotic plaque,anti-hyperlipidemic agents which lower the ratio of LDL-cholesterol toHDL cholesterol are indicated as anti-atherosclerotic agents, hereinincorporated by reference U.S. Pat. Nos. 4,826,876 and 5,466,861.

The present invention also provides a method of producing selectivethyromimetic activity in certain tissues except the heart whichcomprises administering to an animal in need thereof an effective amountto produce said activity of a compound of Formula 1 or apharmaceutically acceptable salt thereof.

The present invention also relates to a method of lowering plasma lipidlevels and a method of lowering the ratio of LDL-cholesterol toHDL-cholesterol levels by suitably administering a compound of thisinvention or a pharmaceutically acceptable sale thereof.

In addition, compounds of Formula 1 may be indicated in thyroid hormonereplacement therapy in patients with compromised cardiac function.

In therapeutic use the compounds of the present invention are usuallyadministered in a standard pharmaceutical composition.

The present invention therefore provides in a further aspectpharmaceutical compositions comprising a compound of Formula 1 or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier. Such compositions include those suitable for oral,parenteral or rectal administration.

Pharmaceutical Compositions

Compounds of Formula 1 and their pharmaceutically acceptable salts whichare active when given orally can be formulated as liquids for examplesyrups, suspensions or emulsions, tablets, capsules and lozenges.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s), for example ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, with a suspendingagent, preservative, surfactant, wetting agent, flavoring or coloringagent. Alternatively, a liquid formulation can be prepared from areconstitutable powder.

For example a powder containing active compound, suspending agent,sucrose and a sweetener can be reconstituted with water to form asuspension; and a syrup can be prepared from a powder containing activeingredient, sucrose and a sweetener.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.Compound of Formula 1 and their pharmaceutically acceptable salts whichare active when given parenterally can be formulated for intramuscularor intravenous administration.

A typical composition for intramuscular administration will consist of asuspension or solution of active ingredient in an oil, for examplearachis oil or sesame oil. A typical composition for intravenousadministration will consist of a sterile isotonic aqueous solutioncontaining, for example active ingredient, dextrose, sodium chloride, aco-solvent, for example polyethylene glycol and, optionally, a chelatingagent, for example ethylenediamine tetracetic acid and an anti-oxidant,for example, sodium metabisulphite. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

Compounds of structure (1) and their pharmaceutically acceptable saltswhich are active on rectal administration can be formulated assuppositories. A typical suppository formulation will generally consistof active ingredient with a binding and/or lubricating agent such as agelatin or cocoa butter or other low melting vegetable or synthetic waxor fat.

Compounds of Formula 1 and their pharmaceutically acceptable salts whichare active on topical administration can be formulated as transdermalcompositions. Such compositions include, for example, a backing, activecompound reservoir, a control membrane, liner and contact adhesive.

The typical daily dose of a compound of Formula 1 varies according toindividual needs, the condition to be treated and with the route ofadministration. Suitable doses are in the general range of from 0.001 to10 mg/kg bodyweight of the recipient per day.

Within this general dosage range, doses can be chosen at which thecompounds of Formula 1 lower plasma cholesterol levels and raisemetabolic rate with little or no direct effect on the heart. In general,but not exclusively, such doses will be in the range of from lower doese(0.001 to 0.5 mg/kg) to higher doses (0.5 to 10 mg/kg).

In addition, within the general dose range, doses can be chosen at whichthe compounds of Formula 1 lower plasma cholesterol levels and havelittle or no effect on the heart without raising metabolic rate. Ingeneral, but not exclusively, such doses will be in the range of from0.001 to 0.5 mg/kg.

It is to be understood that the 2 sub ranges noted above are notmutually exclusive and that the particular activity encountered at aparticular dose will depend on the nature of the compound of Formula 1used.

Preferably, the compound of Formula 1 is in unit dosage form, forexample, a tablet or a capsule so that the patient may self-administer asingle dose. In general, unit doses contain in the range of from0.05-100 mg of a compound of Formula 1. Preferred unit doses containfrom 0.05 to 10 mg of a compound of Formula 1.

The active ingredient may be administered from 1 to 6 times a day. Thusdaily doses are in general in the range of from 0.05 to 600 mg per day.Preferably, daily doses are in the range of from 0.05 to 100 mg per day.Most preferably from 0.05 to 5 mg per day.

EXAMPLES Example 1 Synthesis of TR Ligands

Many TR ligands are known in the art, including T4 (thyroxine), T3, T2and TS-9. See Jorgensen, Thyroid Hormones and Analogs, in 6 HormonalProteins and Peptides, Thyroid Hormones 107-204 (Choh Hao Li ed., 1978),incorporated by reference herein.

The syntheses of several TR ligands are described below.

Synthesis of TS1, TS2, TS3, TS4, TS5

TS1, TS2, TS3, TS4 and TS5 and analogs thereof can all be prepared bysimple acylation of the nitrogen atom of any thyronine analog, includingT3 (3,5,3′-triiodo-L-thyronine), T4 (thyroxine) and 3,5-diiodothyronine.TS1 and TS2 are synthesized by reacting T3 with Ph₂CHCO₂NHS (N-hydroxysuccinimide-2,2-diphenylacetate) and C₁₆H₃₃CO₂NHS, respectively. TS3 issynthesized by reacting T3 with FMOC—Cl(fluorenylmethyloxycarbonylchloride). TS4 is synthesized by reacting T3with tBOC₂O (tBOC anhydride or di-t-butyldicarbonate). TS5, whichdiffers from TS14 by having a —H instead of an —I at the R¹ ₃ position,is synthesized by reacting 3,5-diiodothyronine with tBOC20. The generalreaction scheme for TS1, TS2, TS3, TS4 and TS5 is depicted in FIG. 11.It should be noted that in the reaction scheme, both TS5 and itsprecursor both have a hydrogen rather than an iodine at the R¹ ₃position.

Synthesis of TS6 and TS7

TS6 is synthesized by reacting TS5 with paranitrophenylisocyanate. TS7is synthesized by reacting TS6 with TFA (trifluoroacetic acid), whichcleaves the tBOC group. These reactions are simple organic synthesisreactions that can be performed by anyone of ordinary skill in the art.The synthetic scheme for TS6 and TS7 is diagrammed in FIG. 12.

Synthesis of TS8

TS8 is synthesized by reacting TS5 with Ph₂CHNH₂ (diphenylmethylamine)in the presence of triethylamine and any amide forming condensingreagent, such as TBTU (hydroxybenztriazoleuronium tetrafluoroborate) orHBTU (hydroxybenztriazoleuronium hexafluorophosphate). The synthesisscheme for TS8 is depicted in FIG. 13.

Synthesis of 3,5-Diiodo-3′Isopropylthyronine Derivatives

For designing a class of antagonists, it is important to have ahydrophobic group at the 3′ position as well as an extension at the 5′position. Preferred hydrophobic groups at the 3′ position include:methyl, benzyl, phenyl, iodo, and heterocyclic structures. The synthesisof a 3,5-diiodo-3′-isopropyl-5′-substituted thyronine is describedbelow. The example provided describes the specific steps forsynthesizing the TS10 compound, but this general reaction scheme can beused by one of ordinary skill in the art to synthesize any number of3,5,-diiodo-3′-isopropyl-5′-substituted thyronine derivatives, which arecharacterized by having an extension at the 5′ position. Additionalcompounds of this class can be synthesized using known organic synthesistechniques.

The synthesis of TS10 is described below and is depicted in FIG. 14.Numbers used in the reaction scheme for TS10 indicating the reactionproduct for each step are in parentheses.

2-Formyl-6-isopropylanisole (1): 2-formyl-isopropylanisole (10.0 g, 61mmol), as made by Casiraghi, et al. JCS Perkin I, 1862 (1980)(incorporated by reference), is added dropwise to a suspension of sodiumhydride (3.7 g, 153 mmol) in 50 mL THF and 50 mL of DMF in a roundbottom flask. The addition generates an exothermic reaction andformation of a gray solid. Methyl iodide (26.0 g, 183 mmol) is thenadded dropwise and the reaction mixture is stirred at room temperaturefor 5 hours. The reaction mixture is quenched with mL of water, thenpoured into 500 mL of water, and is extracted with ether (2×300 mL). Theether layers are combined, washed with water (5×1000 mL), dried overmagnesium sulfate and concentrated in vacuo to provide 10.2 g (94%) ofthe title compound, with the following ¹H NMR (CDCl₃) properties: d10.30 (s, 1H), 7.63 (d, 1H, J=3 Hz), 7.50 (d, 1H, J=3 Hz), 7.13 (t, 1H,J=3 Hz), 3.81 (s, 3H), 3.31 (heptet, 1H, J=7.5 Hz), 1.19 (d, 6H, J=7.5Hz).

2-(2-Hydroxynonyl)-6-isopropylanisole (not shown in scheme):Octylmagnesium chloride (8.4 mL, 16.9 mmol, 2.0 M) is added dropwise toa solution of 1 (1.5 g, 8.4 mmol) in 10 mL THF at −78° C. The reactionmixture is stirred for 2 hours with warming to room temperature. Thereaction mixture is diluted with 50 mL ether and poured into 50 mLwater. The ether layer is washed with brine (1×50 mL), dried over sodiumsulfate, and concentrated in vacuo. Flash chromatography (silica gel,10% ether/hexane→15% ether/hexane) provides 734 mg (30%) of the titlecompound with the following ¹H NMR (CDCl₃) properties: d 7.33-7.10 (m,3H), 5.00 (br. s, 1H), 3.81 (s, 3H), 3.33 (heptet, 1H, J=7 Hz) 1.90-1.19(m, 14H), 0.86 (t, 3H, J=6.5 Hz); HRMS (EI), found: 292.2404; calc'd:292.2402.

2-nonyl-6-isopropylanisole (2): Compound 2 (663 mg, 2.3 mmol) isdissolved in solution of 5 mL ethanol and 5 mL acetic acid, and aspatula tip of palladium on carbon catalyst is added. The reactionmixture is then charged with hydrogen gas (using a simple balloon andneedle) and the mixture is stirred at room temperature overnight. Thenext day, the reaction mixture is poured into ether (100 mL) and theether layer is extracted with saturated sodium bicarbonate (3×100 mL).The ether layer is dried over sodium sulfate and concentrated in vacuoto provide 581 mg (91%) of (2) with the following ¹H NMR (CDCl₃)properties: d 7.14-7.00 (m, 3H), 3.75 (s, 3H), 3.36 (heptet, 1H, J=6.8Hz), 2.63 (t, 2H, J=7.5 Hz), 1.68-1.15 (m, 14H), 0.86 (t, 3H, J=5.5 Hz);HRMS (EI), mass found: 276.2459; calculated: 276.2453.

Thyronine adduct (4): Fuming nitric acid (0.071 mL) is added to 0.184 mLacetic anhydride chilled to −5° C. Iodine (66 mg) is added to thismixture followed by trifluoroacetic acid (0.124 mL). This mixture isstirred for 1 hour with warming to room temperature, at which point allof the iodine is dissolved. The reaction mixture was then concentratedin vacuo to provide an oily semi-solid material. The residue wasdissolved in 0.7 mL of acetic anhydride and cooled to −20° C. A solutionof anisole (2) (581 mg, 2.1 mmol) in 1.2 mL acetic anhydride and 0.58 mLTFA is added dropwise. The reaction mixture is stirred at −20° for 1hour, then stirred overnight with warming to room temperature. Thereaction mixture is partitioned between water and methylene chloride.The methylene chloride layer is dried over sodium sulfate andconcentrated in vacuo to provide the iodonium salt (3) as an oil. Thismaterial is not purified or characterized, and is directly introducedinto the coupling reaction.

N-Trifluoroacetyl-3,5-diiodotyrosine methyl ester (552 mg, 1.0 mmol)prepared according to the procedure of N. Lewis and P. Wallbank,Synthesis 1103 (1987) (incorporated by reference) and all of the crudeiodonium salt (3) from above is dissolved in 5 mL of anhydrous methanol.Diazabicyclo[5.4.0]undecane (DBU) (183 mg, 1.2 mmol) and a spatula tipof copper-bronze are added and the resulting mixture is stirred at roomtemperature overnight. The next day, the reaction mixture is filtered,and the filtrate is concentrated in vacuo. The crude residue is purifiedby flash chromatography (silica gel, 10% ethyl acetate/hexane) toprovide 30 mg (4%) of the protected thyronine adduct (4).

Deprotected thyronine (TS10): The protected thyronine 4 (30 mg, 0.04mmol) is dissolved in a mixture of 2.25 mL acetic acid and 2.25 mL 49%hydrobromic acid. The reaction mixture is heated to reflux for 5 hours.The reaction mixture is cooled to room temperature, and the solvents areremoved in vacuo. Water is added to triturate the oily residue into agray solid. This solid material is filtered, washed with water, anddried over P₂O₅ in vacuo to provide 24 mg (81%) of the title compound,TS10, with the following ¹H NMR (CDCl₃) properties: d 7.57 (s, 1H), 6.86(s, 1H), 6.45 (s, 1H), 6.34 (s, 1H), 4.81 (m, 1H), 3.86 (s, 3H), 3.71(s, 3H), 3.33-3.05 (m, 3H), 2.58-2.47 (m, 2H), 1.62-0.76 (m, 23H); MS(LSIMS): M⁺=817.0.

As mentioned above, this reaction scheme can be modified by one ofordinary skill in the art to synthesize a class of compoundscharacterized by 3,5-diiodo-3′isopropylthyronine derivatives, wherein(1) the 3′ isopropyl group can be replaced with a hydrophobic group,including methyl, benzyl, phenyl, iodo, and heterocyclic structures, and(2) a wide variety of chemical structures can be incorporated at the 5′position, including alkyl groups, planar aryl, heterocyclic groups, orpolar and/or charged groups.

The aldehyde (1) in the above reaction scheme is a versatile syntheticintermediate which allows for the attachment of a variety of chemicalmoieties to the 5′ position of the final thyronine derivative. Inaddition, a variety of chemical reactions can be used to attach thechemical moieties. These reactions are well known in the art and includeorganometallic additions to the aldehyde (including Grignard reagents,organolithiums, etc.), reductive amination reactions of the aldehydewith a primary or secondary amine, and Wittig olefination reactions witha phosphorous ylid or stabilized phosphonate anion. Other possibilitiesinclude reduction of the aldehyde to a benzyl alcohol allowing foretherification reactions at the 5′ position. As mentioned above, thesemethods allow for a wide variety of chemical structures to beincorporated at the 5′ position of the final thyronine derivative,including alkyl groups, planar aryl, heterocyclic groups or polar and/orcharged groups.

Synthesis of 3,5-dibromo-4-(3′,5′-diisopropyl-4′-hydroxyphenoxy)benzoicacid (Compound 11)

(a) A mixture of 2,6-diisopropyl phenol (20 g, 0.11 mol), potassiumcarbonate (62 g, 0.45 mol), acetone (160 ml) and methyl iodide (28 ml,0.45 mole) is refluxed for three days. The reaction mixture is filteredthrough celite, evaporated, dissolved in ether, washed twice with 1Msodium hydroxide, dried over magnesium sulphate and concentrated toafford 15.1 g (0.08 mol, 70%) of 2,6-diisopropyl anisole as a slightlyyellow oil.

(b) Fuming nitric acid (12.4 ml, 265 mmol) is added dropwise to 31.4 mlof acetic anhydride which is cooled in a dry ice/carbon tetrachloridebath. Iodine 11.3 g, 44.4 mmol) is added in one portion followed bydropwise addition of trifluoroacetic acid (20.5 ml, 266 mmole). Thereaction mixture is stirred at room temperature until all the iodine isdissolved. Nitrogen oxides are removed by flushing nitrogen into thevessel. The reaction mixture is concentrated, the residue is dissolvedin 126 ml of acetic anhydride and is cooled in a dry ice/carbontetrachloride bath. To the stirred solution 2,6-diisopropylanisole (51g, 266 mmol) in 150 ml of acetic anhydride and 22.6 ml oftrifluoroacetic acid is added dropwise. The reaction mixture is left tostand at room temperature over night and then is concentrated. Theresidue is taken up in 150 ml of methanol and treated with 150 ml of 10%aqueous sodium bisulfite solution and 1 liter of 2M sodiumborotetrafluoride solution. After the precipitate aggregates, petroleumether is added and the supernatant is decanted. The precipitate istriturated with petroleum ether, filtered, washed with petroleum etherand dried at room temperature in vacuo. This affords 34 g (57 mmol, 65%)of bis(3,5-diisopropyl-4-methoxyphenyl)iodonium tetrafluoroborate as awhite solid.

(c) To a stirred solution of 3,5-dibromo-4-hydroxybenzoic acid (12 g,40.5 mmol) in 250 ml of methanol, thionyl chloride (3 ml) is addeddropwise. The reaction mixture is refluxed for five days, water is addedand the precipitated product is filtered off. The residue is dissolvedin ethyl acetate. From the aqueous phase, methanol is removed byconcentration. The aqueous phase is then saturated with sodium chloride,and extracted with ethyl acetate. The combined organic phases are driedover magnesium sulphate, filtered and concentrated. This gives 12.5 g(40.5 mmol, 100%) of 3,5-dibromo-4-hydroxymethyl benzoate as a whitecrystalline solid.

(d) The products obtained in steps b and c are reacted with each otheraccording to the following protocol. Tobis(3,5-diisopropyl-4-methoxyphenyl)iodonium tetrafluoroborate (2.86 g,4.8 mmole) and copper bronze (0.42 g, 6.4 mmole) in 7 ml. ofdichloromethane at 0° C. is added dropwise a solution of3,5-dibromo-4-hydroxymethyl benzoate (1.0 g, 3.2 mmole) andtriethylamine (0.36 g, 3.5 mmole) in 5 ml of dichloromethane. Thereaction mixture is stirred in the dark for eight days and then isfiltered through celite. The filtrate is concentrated and the residue ispurified by column chromatography (silica gel, 97:3 petroleumether/ethyl acetate) to give 0.62 g (1.2 mmole, 39%) of3,5-dibromo-4-(3′,5′-diisopropyl-4′-methoxyphenoxy)methyl benzoate as asolid.

(e) The product from step d (0.2 g, 0.4 mmole) is dissolved in 2 ml.dichloromethane, is put under nitrogen and is cooled at −40° C. To thestirred solution is added 1M BBr₃ (1.2 ml, 1.2 mmole) dropwise. Thereaction mixture is allowed to reach room temperature and then is leftover night. It is cooled to 0° C. and then hydrolyzed with water.Dichloromethane is removed by concentration and the aqueous phase isextracted with ethyl acetate. The organic phase is washed with 1Mhydrochloric acid and brine. Then it is dried over magnesium sulphate,filtered and concentrated. The residue is chromatographed (silica,96:3.6:0.4 dichloromethane/methanol/acetic acid) producing 93 mg (0.2mmole, 51%) of3,5-dibromo-4-(3′,5′-diisopropyl-4′-hydroxyphenoxy)benzoic acid as awhite solid. ¹H nmr (CDCl₃) δ 1.23 (d, 12H, methyl), 3.11 (m, 2H, CH),6.50 (s, 2H, 2,6-H) 8.33 (s, 2H, 2′,6′-H).

Synthesis of addition ligands are described in U.S. Ser. No. 08/877,792,filed Jun. 18, 1997 which is herein incorporated in its entirety byreference.

TABLE 2 and FIG. 15 depict the structures of several TR ligands inreference to Formula I. TABLE 2 Cmpd R₃ R₄ R₅ R′₃ R′₄ R′₅ R₁ *T₃ —I —O——I —I —OH —H —CH₂CH(NH₂)CO₂H *T₄ —I —O— —I —I —OH —I —CH₂CH(NH₂)CO₂H TS1—I —O— —I —I —OH —H —CH₂CH[NHCOCHø₂]CO₂H TS2 —I —O— —I —I —OH —H—CH₂CH[NHCO(CH₂)₁₅CH₃]CO₂H TS3 —I —O— —I —I —OH —H —CH₂CH[NH—FMOC]CO₂HTS4 —I —O— —I —I —OH —H —CH₂CH[NH-tBOC]CO₂H TS5 —I —O— —I —H —OH —H—CH₂CH[NH-tBOC]CO₂H TS6 —I —O— —I —H —OC(O)NH═Ø_(p)NO₂ —H—CH₂CH[NH-tBOC]CO₂H TS7 —I —O— —I —I —OC(O)NH═NHØNO₂ —H —CH₂CH(NH₂)CO₂HTS8 —I —O— —I —H —NH—CHØØ —H —CH₂CH(NH-tBOC]CO₂H TS9 —I —O— —I -IsoPr—OH —H —CH₂CH(NH₂)CO₂H TS10 —I —O— —I -IsoPr —OH —(CH)₈— —CH₂CH(NH₂)CO₂HCH₃*Prior Art Compound-Ø: phenyl-ØpNO₂: para nitro phenyl

Example 2 Receptor Binding Assays of TR Ligands

To test the ability of synthesized TR ligands to bind to a thyroidreceptor (TR), the binding affinity of a TR ligand for TR is assayedusing TR's prepared from rat liver nuclei and 125₁ T₃ as described in J.D. Apriletti, J. B. Baxter, and T. N. Lavin, J. Biol. Chem., 263:9409-9417 (1988). The apparent Kd's are calculated using the methoddescribed by Apriletti (1995) and Apriletti (1988). The apparent Kd'sare presented in TABLE 3. The apparent Kd's (App.Kd) are determined inthe presence of the sample to be assayed, 1 nM [¹²⁵I]T₃, and 50 μg/mlcore histones, in buffer E (400 mM KCl, 200 mM potassium phosphate, pH8.0, 0.5 mM EDTA, 1 mM MgCl₂, 10% glycerol, 1 mM DTT) in a volume of0.21 ml. After incubation overnight at 4° C., 0.2 ml of the incubationmixture is loaded onto a Quick-Sep Sephadex G-25 column (2.7×0.9 cm, 1.7ml bed volume) equilibrated with buffer E. The excluded peak ofprotein-bound [¹²⁵I]T₃ is eluted with 1 ml of buffer E, collected in atest tube, and counted. Specific T₃ binding is calculated by subtractingnonspecific binding from total binding. TABLE 3 Coactivation AssayCompound App.Kd(nM) RIP-140 EC₅₀(M) T₃ 0.06 + 10⁻¹⁰ T₄ 2 + 10⁻⁹  TS1 4 +10⁻⁷  TS2 1400 nd nd TS3 4 + 10⁻⁸  TS4 8 + nd TS5 220 + 10⁻⁶  TS6 >10000nd nd TS7 260 + 10⁻⁷  TS8 6000 nd nd TS9 1 + 10⁻¹⁰ TS10 400 + 10⁻⁶ +: RIP-140 Binding−: RIP-140 Bindingnd: Not Determined

Example 3 Increased Nuclear Protein Coactivation by TR Ligands

To test the ability of TR ligands to activate the binding of TR to thenuclear activation protein RIP-140 (a nuclear protein that can bind tonuclear receptors, such as the estrogen receptor), a TR ligand isliganded to TR and then incubated with RIP-140 as described in V.Cavailles, et al., EMBO J., 14(15):3741-3751 (1995), which isincorporated by reference herein. In this assay, 35_(S)-RIP-140 proteinbinds to liganded TR but not unliganded TR. Many TR 35_(S) ligands canactivate RIP-140 binding as shown in TABLE 3.

Example 4 TR Ligand Binding and TR Activation in Cultured Cells

To test TR activation of transcription in a cellular environment, TRligands are assayed for their ability to activate a reporter gene,chloramphenicol transferase (“CAT”), which has a TR DNA binding sequenceoperatively linked to it. Either GC or L937 cells (available from theATCC) can be used, respectively). In such assays, a TR ligand crossesthe cell membrane, binds to the TR, and activates the TR, which in turnactivates gene transcription of the CAT by binding the TR DNA bindingregion upstream of the CAT gene. The effective concentration for halfmaximal gene activation (EC₅₀) is determined by assaying CAT geneactivation at various concentrations as described herein and in theliterature. The results of CAT gene activation experiments are shown inTABLE 3.

CAT Gene Activation Assays

Functional response to thyroid hormone (3,5,3′-triiodo-L-thyronine, T₃)and TR ligands is assessed either in a rat pituitary cell line, GCcells, that contain endogenous thyroid hormone receptors (TRs) or U937cells that contain exogenous TRs expressed as known in the art. GC cellsare grown in 10-cm dishes in RPMI 1640 with 10% newborn bovine serum, 2mM glutamine, 50 units/ml penicillin and 50 μg/ml streptomycin. Fortransfections, cells are trypsinized, resuspended in buffer (PBS, 0.1%glucose) and mixed with a TREtkCAT plasmid (10 mg) or phage in 0.5 mlbuffer (15±5 million cells) and electroporated using a Bio-Rad genepulser at 0.33 kvolts and 960 mF. The TREtkCAT plasmid contains twocopies of a T₃ response element (AGGTCAcaggAGGTCA) cloned in the HindIII site of the pUC19 polylinker immediately upstream of a minimal(−32/+45) thymidine kinase promoter linked to CAT (tkCAT) codingsequences. After electroporation, cells are pooled in growth medium(RPMI with 10% charcoal-treated, hormone stripped, newborn bovineserum), plated in 6-well dishes and treated with either ethanol orhormone. CAT activity is determined 24 hours later as described D. C.Leitman, R. C. J. Ribeiro, E. R. Mackow, J. D. Baxter, B. L. West, J.Biol. Chem. 266, 9343 (1991), which is incorporated by reference herein.

Effect of TS-10 on the Transcriptional Regulation of the DR4-ALPReporter Gene in the Presence or Absence of T3.

Characteristics of the TRAF cells: TRAFa1 are CHO K1 cells stablytransformed with an expression vector encoding the human thyroid hormonereceptor α1 and a DR4, ALP reporter vector; TRAFb1 are CHO K1 cellsstably transformed with an expression vector encoding the human thyroidhormone receptor β1 and a DR4-ALP reporter vector.

Interpretation of the Effect of Compound TS-10 on the TranscriptionalRegulation of the DR4-ALP Reporter Gene in the Presence or Absence ofT3.

TRAFa1 reporter cells: TS-10 alone (open circles) induces a partialactivation of the expression of the ALP reporter protein amounting toapproximately 27% of the maximal effect by the natural thyroid hormoneT3. In the presence of T3 (filled circles), TS-10 has a weakantagonistic effect. The EC50 concentration for the agonistic effect ofTS-10 and the EC50 concentration for its T3 antagonistic effect,respectively, is indicated in FIG. 18.

In FIG. 18, open and filled circles with dotted lines show thedose-dependent effect of TS-10/T3 on the toxicity marker MTS/PMS),reduction of tetrazolium salt in the mitochondria, displayed on theright y-axis as optical density. There is no obvious toxic effect ofTS-10 on the MTS-PMS marker but there is a clear effect on themorphology of the cells, as can be seen under the light microscope, atthe highest concentration of TS-10 (32 mM) both in the absence andpresence of T3, respectively (not shown in the figure).

TRAFb1 reporter cells: TS-10 alone (open circles) induces a partialactivation of the expression of the ALP reporter protein amounting toapproximately 35% of the maximal effect by T3. The EC50 concentrationfor the agonistic effect of TS-10 is indicated in FIG. 19. In thepresence of T3 (filled circles), TS-10 shows, if anything, a slightpotentiation of the T3 effect on the expression of the ALP reporterprotein. The T3 inhibitory effect of TS-10 at its highest concentrationused (32 mM) is a toxic effect rather than T3 antagonism.

In FIG. 19, open and filled circles with dotted lines show thedose-dependent effect of TS-10/T3 on the toxicity marker (MTS/PMS),reduction of tetrazolium salt in the mitochondria, displayed on theright y-axis as optical density. There is no obvious toxic effect ofTS-10 on the MTS-PMS marker but a clear effect on the morphology of thecells can be observed, under the light microscope, at the highestconcentration of TS-10 (32 mM) both in the absence and presence of T3,respectively (not shown in the figure).

HepG2 (HAF18) reporter cells: TS-10 alone (open circles) induces apartial activation of the expression of the ALP reporter proteinamounting to slightly more than 50% of the maximal effect by T3. TheEC50 concentration for the agonistic effect of TS-10 is indicated inFIG. 20. In the presence of T3 (filled circles), TS-10 shows no effecti.e. no T3 antagonism nor potentiation/additive effect to T3. Open andfilled circles with dotted lines show the dose-dependent effect ofTS-10/T3 on the toxicity marker (MTS/PMS), reduction of tetrazolium saltin the mitochondria, displayed on the right y-axis as optical density.There is no obvious toxic effect of TS-10 on the MTS/PMS marker or onthe morphology of the cells, as can be observed using a lightmicroscope, at any concentration of TS-10/T3 used.

Example 5 Comparisons of Human TR-α and Human TR-β

Competition for [¹²⁵I]T₃ Binding to TR LBD by T₃ and Triac

The drug, Triac, is a thyroid hormone agonist. Triac is3,5,3′-triiodothyroacetic acid and is described in Jorgensen, ThyroidHormones and Analogs in 6 Hormonal Proteins and Peptides, ThyroidHormones at 150-151 (1978). Another compound that can be used in placeof Triac is 3,5-diiodo-3′-isopropylthyroacetic acid. Competition assaysare performed to compare the displacement of [¹²⁵I]T₃ from binding withhuman TR-α LBD or human TR-β LBD by unlabeled T₃ or Triac. The resultsof such assays are depicted in FIG. 16.

Standard binding reactions are prepared containing 1 nM [¹²⁵I]T₃, 30fmol of human TR-α (empty symbols) or β (solid symbols), and variousconcentrations of competing unlabeled T₃ (circles) or Triac (triangles).Assays are performed in duplicate.

Competition for [¹²⁵I]T₃ Binding to Variant TR LBD by T₃, Triac and GC-1

The following assays residues involved in selective binding among TRisoforms. Competition assays are performed to compare the displacementof [¹²⁵I]T₃ from binding with wild-type human TR-α LBD or human TR-βLBD, to a variant form of the TR LBDs by unlabeled T₃, Triac or GC-1. Avariant TR-α or TR-β is constructed by substituting an amino acid foundin the corresponding position of the other TR isoform. For example,asparagine 331 in human TR-α corresponds to serine 277 in human TR-α. Totest binding specificity contributed by this position, a variant humanTR-β is constructed that contains asparagine 331 substituted with aserine residue (designated Asn331Ser or N331S). Binding assays aredescribed in Apriletti et al. (Protein Expression and Purification6:363-370 (1995)). The results of such assays are depicted in FIG. 27,and summarized in Table 4 below. TABLE 4 Effect of TR-β SubstitutionN331S on Binding Affinity Ligand Native TR-α Native TR-β Mutant TR-β T320 pM 60 pM 100 pM T4 600 3000 ND Triac 20 20 100 IpBr₂ 17 ND ND Dimit6000 8000 ND GC-1 200 40 400Competition curves comparing wildtype TR-β versus the variant TR-β N331Sfor binding T3, Triac or GC-1 show that the affinity of the mutantreceptor for Triac was reduced to approximately the same as for T3 (vs.3-fold greater in wild type) so that the relative affinities are similarto wild-type TR-α. The affinity for GC-1 was also reduced to severalfold less than T3, as is seen with TR-α.

Comparison of the affinity of TR-β variant N331S to the native TRs forselected ligands is as follows:

Native TR-α for various ligands (T3, T4, Triac, IpBr2, Dimit, GC-1):

-   -   IpBr₂>Triac≅T3>GC-1>T4>Dimit

Native TR-β (T3, T4, Triac, Dimit, GC-1)

-   -   Triac>GC-1≧T3>T4>Dimit

Variant TR-β (N331S) (T3, Triac, GC-1)

-   -   Triac≅T3>GC-1.

Scatchard Analysis of [¹²⁵I]T3 Binding to TR

Human TR-α (left panel) or human TR-β (right panel) is assayed for T₃binding in the presence of increasing concentrations of [¹²⁵I]T₃. Theapparent equilibrium dissociation constant (20 pM for α and 67 pM for β)is calculated by linear regression analysis and is depicted in FIG. 17.

3,5-dibromo-4-(3′,5′-diisopropyl-4′-hydroxyphenoxy)Benzoic Acid is aTR-α Selective Synthetic Ligand.

3,5-dibromo-4-(3′,5′-diisopropyl-4′-hydroxyphenoxy)benzoic acid(Compound 11), the structure of which is drawn above, is assayed forbinding to the two different isoforms of the TR, TR-α and TR-β. Compound11 exhibits an IC50 of 1.6 μM for binding to TR-α and an IC50 of 0.91 μMfor binding to TR-β. Assays for determining selective binding to theTR-α or TR-β LBD can include reporter assays, as described herein. Seealso Hollenberg, et al., J. Biol. Chem., (1995) 270(24):14274-14280.

Example 6 Preparation and Purification of a TR-α LBD

Rat TR-α LBD, residues Met122-Val410, is purified from E. coli(“LBD-122/410”). The expression vector encoding the rat TR-α LBD isfreshly transfected into E. coli strain BL21(DE3) and grown at 22° C. ina 50-liter fermenter using 2×LB medium. At an A₆₀₀ of 2.5-3, IPTG isadded to 0.5 mM and growth is continued for 3 h before harvesting. Thebacterial pellet is quickly frozen in liquid nitrogen and stored at −70°C. until processed. Extraction and purification steps are carried out at4° C. The bacteria are thawed in extraction buffer (20 MM Hepes, pH 8.-,1 mM EDTA, 0.1% MTG, 0.1 mM PMSF, and 10% glycerol) at a ratio of 10 mlbuffer/g bacteria. Bacteria are lysed by incubation for 15 min. with 0.2mg/ml lysozyme and sonicated at maximum power while simultaneouslyhomogenized with a Brinkmann homogenizer (Model PT 10/35 with generatorPTA 35/2) until the solution loses its viscosity. After centrifugationfor 10 min at 10,000 g, the supernatant is adjusted to 0.4 M KCl,treated with 0.6% PEI to precipitate fragmented DNA, and centrifuged for10 min at 10,000 g. The rat TR-α LBD in the supernatant is thenprecipitated with 50% ammonium sulfate and centrifuged for 10 min at10,000 g. The precipitate is resuspended with buffer B (20 mM Hepes, pH8.0, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 0.01% Lubrol, and 10% glycerol)to a final conductivity of 9 mS/cm (approx. 0.7 M ammonium sulfate) andcentrifuged 1 h at 100,000 g. The supernatant is frozen in liquidnitrogen and stored at −70° C.

The crude extract is thawed, bound with a tracer amount of [¹²⁵I]T₃, andloaded directly onto a phenyl-Toyopearl hydrophobic interaction column(2.6×18 cm, 95 ml bed volume) at 1.5 ml/min. The column is eluted with a2-h gradient from 0.7 ammonium sulfate, no glycerol to no salt, 20%glycerol in buffer C (20 mM Hepes, pH 8.0, 0.5 mM EDTA, 1 mM DTT, 0.2 mMPMSF). The rat TR-α LBD prebound to tracer [¹²⁵I]T₃ (less than 0.005% oftotal rat TR-α LBD) is detected using a flow-through gamma emissiondetector, whereas unliganded rat TR-α LBD is assayed by postcolumn[¹²⁵I]T₃ binding assays (described herein).

The phenyl-Toyopearl unliganded rat TR-α LBD peak fractions are pooled,diluted with buffer B to a conductivity of 0.5 mS/cm (equivalent toapprox. 20 mM ammonium sulfate), loaded onto a TSK-DEAE anion-exchangecolumn (2×15 cm, 47 ml bed volume) at 4 ml/min, and eluted with a 60-mingradient from 50 to 200 mM NaCl in buffer B.

The unliganded rat TR-α LBD peak fractions from TSK-DEAE are pooled,diluted twofold with buffer B; loaded at 0.75 ml/min on a TSK-heparinHPLC column (0.8×7.5 cm, 3 ml bed volume), and eluted with a 50 to 400mM NaCl gradient in buffer B.

The pool of unliganded rat TR-α LBD peak fractions from the TSK-heparincolumn is adjusted to 0.7 M ammonium sulfate, loaded at 0.75 ml/min on aTSK-phenyl HPLC column (0.8×7.5 cm, 3 ml bed volume), and eluted with a60-min gradient from 0.7 M ammonium sulfate without glycerol to no saltwith 20% glycerol in buffer C. The fractions containing unliganded ratTR-α LBD are pooled and incubated with a five fold excess of hormone for1 h, the salt concentration is adjusted to 0.7 M ammonium sulfate, andthe sample is reloaded and chromatographed on the same column asdescribed above.

Example 7 Crystallization of Liganded TR-α LBD

Material from a single LBD-122/410 preparation is divided into batches,and quantitatively bound with one of the following ligands: Dimit, T₃,or Triac IpBr₂ (3,5dibromo-3′isopropylthyronine) for the finalpurification step.

To maintain full saturation of rat TR-α LBD with a ligand, and toprepare the complex for crystallization, the ligand-bound rat TR-α LBDis concentrated and desalted in an Amicon Centricon-10 microconcentrator(McGrath et al, Biotechniques, (1989) 7:246-247, incorporated byreference herein), using 10 mM Hepes (pH 7.0), 3.0 mM DTT, and 1.0 nM to10 nM ligand.

Factorial crystallization screening trials (Jancarik & Kim, J. Appl.Crystallogr. (1991) 24:409-411, incorporated by reference herein) arecarried out for rat TR-α LBD bound to selected ligands usinghanging-drop vapor diffusion at 17° C. (with 1 μl protein solution, 1 μlprecipitant solution and a 0.5 ml reservoir using silanized coverslip:(McPherson, Preparation and Analysis of Protein Crystals (1982),incorporated by reference herein). Rat TR-α LBD is not stable at 4° C.and is stored at −80° C., where it maintains its avidity for hormone andits crystallizability for approximately two to three months. Theseprocedures are carried out as described in McGrath, M. E. et al., J.Mol. Biol. (1994) 237:236-239 (incorporated by reference). Crystals areobtained in condition 21 of the screening trials (Jancarik & Kim 1991)and conditions are then optimized. Wedge-shaped crystals arereproducibly obtained with hanging-drop vapor fusion at 22° C. with 15%2-methyl-2,4-pentanediol (MPD), 0.2 M ammonium acetate and 0.1 M sodiumcacodylate (pH 6.7), 3 mM DTT, with 2 μl protein solution, 1 μlprecipitant solution and a 0.6 ml reservoir using silanized coverslip,and with 8.7 mg/ml (Dimit), 5.5 mg/ml (IpBr2), 5 mg/ml (Triac), or 2.3mg/ml (T₃) over a period of three days. Under these conditions,diffraction quality crystals (dimension 0.5×0.2×0.0075 mm³) can be grownat ambient temperature (22° C.). The best crystals have a limitingdimension of approximately 100 μm and are obtained at a proteinconcentration between 2.3 and 8.7 mg/ml in the presence of 3 mM DTT. Thecrystals are of the monoclinic space group C2, with one monomer in theasymmetric unit.

Example 8 Crystallization of Human TR-β LBD Complexed with T3, Triac, orGC-1

Human TR-β LBD complexed with T₃, Triac, or GC-1 are purified accordingto the same procedures described above for the rat TR-α LBD, with thefollowing modifications.

The expression of human TR-β LBD differs from the rat TR-α LBD in thatthe human TR-β LBD residues extend from the amino acid at position 716through the amino acid at position 1022, according to the amino acidnumbering scheme for the various nuclear receptor LBDs depicted in FIG.3. FIG. 3 illustrates a numbering scheme applicable to all of thenuclear receptors listed as well as to any additional homologous nuclearreceptors. The vertical lines on FIG. 3 at position 725 and at position1025 delineate the preferred minimum amino acid sequence necessary toobtain adequate binding of ligand. The amino acid sequence from position716 to position 1022 according to the numbering scheme of FIG. 3corresponds to the amino acid positions 202 to 461 according to theconventional numbering of the amino acid sequence of human TR-β which ispublicly available. Also, the human TR-β LBD is expressed with ahistidine tag, as described in Crowe et al., Methods in MolecularBiology (1994) 31:371-387, incorporated by reference herein.

The purification of human TR-β LBD is the same as that described abovefor the rat TR-α LBD with the following exceptions. First, before thepurification step using the hydrophobic interaction column, a step isadded in which the expressed human TR-β LBD is purified using a nickelNTA column (commercially available from Qiagen, Chatsworth, Calif.)according to manufacturer's instructions, and eluted with 200 mMimidazole. The second difference is that in the purification of thehuman TR-β LBD, the purification step using a heparin column is omitted.

The crystallization of human TR-β LBD bound to T₃, Triac or GC-1 is asfollows. Crystals are obtained in condition 7 of the factorial screenusing hanging drops as before at ambient temperature (22° C.) using thefactorial crystallization screening trials of Jancarik & Kim (1991) andusing the commercially available product from Hampton Research,Riverside). The following are optimum conditions: hexagonal bipyrimidalcrystals are grown at 4° C. for 2-3 days from hanging drops containing1.0-1.2 M sodium acetate (pH unadjusted) and 0.1 M sodium cacodylate (pH7.4), 3 mM DTT, with either a 1 μl protein solution, 1 μl precipitantsolution or 2 μl protein solution, 1 μl precipitant solution and a 0.6ml reservoir using silanized coverslip, at a protein concentration of7-10 mg/ml. The best crystals have a limiting dimension of 200 μm. Thefollowing are optimum conditions for crystallization of the TR-β LBDwith GC-1: hexagonal bipyrimidal crystals are grown at 4° C. for 2-3days from hanging drops containing 0.8-1.0M sodium acetate (pHunadjusted), 50-200 nM sodium succinate, and 0.1M sodium cacodylate (pH7.2), 3 mM DTT, 1 μl protein solution, 1 μl precipitant solution and a0.6 ml reservoir using silanized coverslip, at a protein concentrationof 7-10 mg/ml. The best crystals have a limiting dimension of 200 μM.The unit cell dimensions are cell length a=b=68.73, cell lengthc=130.09. The unit cell angles are α=90°, β=90°, γ=120°.

The crystal system for human TR-β LBD bound to T₃, Triac or GC-1 istrigonal with the space group p3₁21. The unit cell dimensions are celllength a=cell length b=68.448 angstroms, cell length c=130.559angstroms. The angles are α=90°, β=90°, gamma=120°.

Example 9 Determination of Liganded TR-α LBD and TR-β Crystal Structures

Data from each cocrystal (Rat TR-α LBD with Dimit, T3 and IpBr2; HumanTR-β LBD with Triac and GC-1) is measured on a Mar area detector atStanford Synchrotron Radiation Laboratory beamline 7-1 (λ=1.08angstroms) using 1.2° oscillations. Data from the cocrystal of the hTR-βLBD with Triac is measured on a Mar area detector at StanfordSynchrotron Radiations Laboratory beamline 7-1 (λ=1.08 angstroms) using1.0 oscillations. Data from the cocrystal of the hTR-β LBd with GC-1 ismeasured on a R-axis II area detector on a Rigaku rotating Cu anode (50kV, 300 mA). The crystals are transferred into a cryosolvent containing1.2M sodium acetate, 0.1M sodium cacodylate, adn 15% glycerol followedby a second transfer into 30% glycerol, then flash frozen in liquidnitrogen. An orientation matrix for each crystal is obtained usingDENZO. The reflections are integrated with DENZO (commercially availablefrom Molecular Structure Corp., The Woodlands, Tex.) and are scaled withSCALEPACK (as described in Otwinowski, Z, Proceedings of the CCP4 StudyWeekend: “Data Collection and Processing,” 56-62 (SERC DaresburyLaboratory, Warrington, UK 1993) incorporated by reference).

For rTR-α cocrystals, data from the T₃ cocrystal is measured with the b*axis approximately parallel with the spindle. The crystals are flashfrozen at −178° C. in a nitrogen gas stream with the MPD mother liquorserving as the cryosolvent. An orientation matrix for each crystal isdetermined using REFIX (Kabsch, W., J. Appl. Crystallogr. (1993)26:795-800 incorporated by reference). Reflections are integrated withDENZO, and are scaled with SCALEPACK.

For the T₃ data set, Bijvoet pairs are kept separate, and are locallyscaled using MADSYS (W. Hendrickson (Columbia University) and W. Weis(Stanford University)).

Cocrystals prepared from the three isosteric ligands are isomorphous.MIR analysis is performed using programs from the CCP4 suite(Collaborative Computational Project, N. R. Acta Crystallogr. (1994)D50:760-763, incorporated by reference herein). Difference Pattersons iscalculated for both T₃ and IpBr₂, taking the Dimit cocrystal as theparent. The positions of the three iodine atoms in the T₃ differencePatterson are unambiguously determined from the Harker section of thedensity map as peaks of 11σ above background. The positions for the twobromine atoms in the IpBr₂ cocrystals, are located independently, aspeaks 8σ above the noise level. Phases for the LBD-122/410 arecalculated from the solution to the IpBr₂ difference Patterson, and areused to confirm the location of the unique third iodine of the T₃cocrystal. Halogen positions are refined with MLPHARE, including theanomalous contributions from the iodine atoms (Otwinowski, Z.Proceedings of the CCPR Study Weekend 80-86 (SERC Daresbury Laboratory,Warrington, UK 1991)). The MIRAS phases are improved through solventflattening/histogram matching using DM (Cowtan, K., Joint CCP4 andESF-EACBM Newsletter on Protein Crystallography (1994) 31: 34-38,incorporated by reference herein).

A model of the LBD-122/410 with Dimit bound is built with the program 0from the solvent flattened MIRAS 2.5 angstrom electron density map(Jones et al., Acta Crystallogr. (1991) A 47:110-119, incorporated byreference herein). The initial model, without ligand, (Rcryst=40.1%), isrefined using least-squares protocols with XPLOR. The Dimit ligand isbuilt into unambiguous Fo−Fc difference density during the followinground. Subsequent refinement employs both least-squares and simulatedannealing protocols with XPLOR (Brunger et al., Science (1987)235:458-460), incorporated by reference herein). Individual atomicB-factors are refined isotropically. As defined in PROCHECK, allresidues are in allowed main-chain torsion angle regions as described inLaskowski et al., J. Appl. Crystallogr., (1993) 26:283-291, incorporatedby reference herein. The current model is missing 34 residues(Met₁₂₂-Gln₁₅₆) at the N-terminus, and 5 residues (Glu₄₀₆-Val₄₁₀) at theC-terminus.

In addition, the following residues are not modeled beyond Cβ due topoor density: 184, 186, 190, 198, 206, 209, 240, 301, 330, 337, 340,343, 359, and 395. The average B-value for protein atoms is 34.5 Å². Thefinal model consists of the LBD-122/410, residues Arg₁₅₇-Ser₁₈₃,Trp₁₈₅-Gly₁₉₇, Ser₁₉₉-Asp₂₀₆ and Asp₂₀₈-Phe₄₀₅; threecacodylate-modified cysteines: Cys₃₃₄, Cys₃₈₀ and Cys₃₉₂; and 73 solventmolecules modeled as water (2003 atoms).

*R_(sym)=100×Σ_(hkl)Σ_(i)|I_(i)−I|/Σ_(hkl)Σ_(i)I_(i)

†R_(der)=100×Σ_(hkl)|F_(PH)−F_(H)|/Σ_(hkl)|F_(P)|

The occupancy for the two bromine sites is set to 35 electrons. Theoccupancies of the iodine sites are relative to this value.

§Phasing power=

FH

/

ε

, where

FH

is the mean calculated heavy atom structure factor amplitude and

ε

is the mean estimated lack of closure.

∥Rcullis=

ε

/

iso

, where

ε

is the mean estimated lack of closure and

iso

is the isomorphous difference.

¶Rcryst=100×Σ_(hkl)|F_(o)−Fc|/Σ_(hkl)|F_(o)| where F_(o) and F_(c) arethe observed and calculated structure factor amplitudes (for dataF/σ>2). The Rfree was calculated using 3% of the data, chosen randomly,and omitted from the refinement.

§ Correlationcoefficient=Σ_(hkl)(|F_(o)|−|F_(o)|×(|F_(c)|−|F_(c)|)/Σ_(hkl)(|F_(o)|−|F_(o)|)²×Σ_(hkl)(|F_(c)|−|F_(c)|)²

Example 10 Phasing of the rTR-α LBD and hTR-β LBD Complex with Triac

Due to the possible non-isomorphism of the rTRα LBD complex with Triac,a molecular replacement solution is determined using AMORE (Navaza, J.,Acta Crystallographica Section A—Fundamentals of Crystallography (1994)50:157-63 from a starting model consisting of rTRα LBD complex with T₃,but with the ligand, all water molecules, and the following residuesomitted: Asn 179, Arg228, Arg262, Arg266, and Ser 277. Strong peaks areobtained in both the rotation and translation searches, with nosignificant (>0.5 times the top peak) false solutions observed (Table6). Strong positive density present in both the anomalous andconventional difference Fourier maps confirm the solution. Maps arecalculated using sigma-A weighted coefficients output by REFMAC(Murshudov, et al. “Application of Maximum Likelihood Refinements,” inRefinement of Protein Structures, Proceedings of Daresbury Study Weekend(1996)) after 15 cycles of maximum likelihood refinement. Triac, theomitted residues, and water molecules 503, 504, 534 (following thenumbering convention for the TR complex with T3) are built into theresulting difference density using O (Jones et. al.); the conformationsof these residues are further confirmed in a simulated-annealing omitmap (Brunger et. al.). The complete model is then refined usingpositional least-squares, simulated annealing, and restrained, grouped Bfactor refinement in XPLOR to an Rcryst of 23.6% and an Rfree of 24.1%

Phasing of a related LBD using the structure of the rTR-α LBD isconducted as follows. A molecular replacement solution for the hTR-β LBDcomplex with Triac is determined using AMORE from a starting modelconsisting of the rTR-α LBD complexed with T3, but with the ligand andall water molecules omitted. Strong peaks are obtained in both therotation and translation searches, with no significant (>0.5 times thetop peak) false solutions (Table 7). Strong positive density present inboth the anomalous and conventional difference Fourier maps confirm thesolution. Initial maps are calulated using sigma-A weighted coefficientsoutput by REFMAC after 9 cycles of maximum likelihood refinement. Thereal-space fit for each residues was calculated using OOPS (Kleywegt, GJ and Jones, T A, OOPS-a-daisy, ESF/CCP4 Newsletter 30, June 1994, pp.20-24) and the residues with a real-space fit less than 2 standarddeviations below the mean removed: Ala253-Lys263; Glu245-Leu250. Toreduce bias, the following residues were modeled as alanine: Arg282,Arg316, Arg 320, Asn 331. Cycles of rebuilding and positionalleast-squares, simulated annealing, and restrained, grouped B factorrefinement with XPLOR produce a model with an R_(cryst) of 25.3 and anR_(free) of 28.9%. The final model consists of hTR-β LBD residuesGlu202-Gln252, Val264-Glu460; three cacodylate-modified cysteines withthe cacodylate moeity modeled as free arsenic: Cys294, Cys298, Cys388,and Cys434; and 35 solvent molecules modeled as water.

Example 11 Connecting QSAR with Structure in the Thyroid HormoneReceptor

The conclusions of classic thyroid hormone receptor quantitativestructure-activity relationships may be summarized as follows:

1) the R₄′-hydroxyl group functions as a hydrogen bond donor;

2) the amino-propionic acid interacts electrostatically through thecarboxylate anion with a positively charged residue from the receptor;

3) the preferences of R₃/R₅ substituent are I>Br>Me>>H;

4) the preferences of the R₃′-substituent are Ipr>I>Br>Me>>H.

The structure of the thyroid hormone receptor ligand binding domaincomplexed with the agonists T3, IpBr₂, Dimit, Triac, and GC1 as providedherein, permits:

-   -   1) the identification of receptor determinants of binding at the        level of the hydrogen bond;    -   2) the association of these determinants with the predictions of        classic thyroid hormone receptor QSAR; and    -   3) prediction as to which determinants of binding are rigid, and        which are flexible, for both the ligand and the receptor.        This classification for the agonists of the type        (R₁=amino-propionic, acetic acid; R₃, R₅=I, Br, Me; R₃′=Ipr, I)        is given below (for the representative ligand T₃);        F=Fiducial (always satisfied)        A=Adjustable

Based upon the methods and data described herein, the following is anembodiment of the computational methods of the invention, which permitdesign of nuclear receptor ligands based upon interactions between thestructure of the amino acid residues of the receptor LBD and the fourdifferent ligands described herein. The small molecule structures forthe ligands can be obtained from Cambridge Structural Database (CSD),and three dimensional models can be constructed using the methodsdescribed throughout the specification. The following are factors toconsider in designing synthetic ligands:

1) Histidine 381 acts as a hydrogen bond acceptor for the R₄′ hydroxyl,with the optimal tautomer maintained by water molecules. See FIG. 23 andFIG. 24. Histidine is the only hydrophilic residue in this hydrophobicpocket that surrounds the R₄′ substituent. Histidine can be either ahydrogen bond acceptor or donor, depending on its tautomeric state. Itis preferably a hydrogen bond donor, but can tolerate being a hydrogenbond acceptor, as for example, when there is a methoxy at the R₄′position of the ligand;

2) Arginines 228, 262, and 266 interact directly and throughwater-mediated hydrogen bonds with the R₁-substituent, with theelectrostatic interaction provided by Arginine 266 (as in the Triaccomplex). This polar pocket is illustrated by FIG. 23-FIG. 25. FIG. 23depicts T₃ in the TRα ligand binding cavity, where T3's amino-propionicR1-substituent interacts with Arg 228, HOH5O2, H9H5O3 and HOH5O4 viahydrogen bonds. FIG. 24 depicts Triac in the ligand binding cavity, withits —COOH R₁ substituent in the polar pocket. In FIG. 24, Arg 228 nolonger shares a hydrogen bond with the ligand, but the —COOH R₁substituent forms hydrogen bonds with Arg 266. FIG. 25 superimposes T₃and Triac in the ligand binding cavity and shows several positionallyunchanged amino acids and water molecules, and selected changedinteracting amino acids and water molecules. The three figuresillustrate parts of the polar pocket that can change and those partsthat do not move upon binding of different ligands. For example, the Arg262 at the top of the polar pocket does not move, even when the R₁substituent has changed from a —COOH to an aminopropionic acid group.However, the other two Arginines, Arg 228 and Arg 266, demonstrateflexibility in the polar pocket to respond to the change in the size orchemical naure of the R₁ substituent.

3) Inner and outer pockets for the R₃/R₅ substituents are formed bySer260, Ala263, Ile299; and Phe 218, Ile221, Ile222, respectively. SeeFIGS. 21 and 22. The inner pocket is filled by either the R₃ or the R₅substituent, regardless of the size of the substituent, and may act as abinding determinant by positioning the ligand in the receptor.Optimally, the inner pocket amino acids interact with an R3 or R5substituent that is no larger than an iodo group. If the inner pocket isfilled by the R₃ substituent, then the outer pocket interacts with theR₅ substituent and vice versa. The outer pocket can adjust to the sizeof its substituent through main chain motion centered at the break inhelix 3 (Lys220-Ile221), suggesting that the bending of H3, and motionof the N-terminal portion of H3, may represent a conformational changeinduced on ligand binding. The outer pocket has greater flexibility thandoes the inner pocket in terms of accommodating a larger substituentgroup.

4) A pocket for the R₃′-substituent is formed by Phe 215, Gly290,Met388. The pocket is incompletely filled by the R₃′-iodo substituent,and accommodates the slightly larger 3′-isopropyl substituent bymovement of the flexible Met388 side chain and the H7/H8 loop. Thispocket can accommodate R₃′ substituents that are even larger thanisopropyl, for example, a phenyl group.

The above information will facilitate the design of high affinityagonists and antagonists by improving automated QSAR methodologies andinforming manual modeling of pharmaceutical lead compounds. For example,the inclusion of discrete water molecules provides a completedescription of hydrogen bonding in the polar pocket for use withpharmacophore development: also, the identification of mobile andimmobile residues within the receptor suggests physically reasonableconstraints for use in molecular mechanics/dynamics calculations.

Example 12 Design of an Increased Affinity Ligand

Direct interaction between the receptor and the ligand is limited in thepolar pocket, which interacts with the R₁ substituent. While the lack ofcomplementarity may contain implications for biological regulation, italso provides an opportunity for increasing affinity by optimizing theinteraction between the amino acids of the polar pocket and the R₁substituent of a synthetic ligand. The structure of the receptor-ligandinteractions described herein enables design of an increased affinitysynthetic ligand having two complementary modifications:

1) Remove the positively charged amine. The strongly positiveelectrostatic potential predicted for the polar pocket suggests that thepositively charged amine of the aminopropionic acid R₁ substituent maybe detrimental to binding. Suitable groups for substitution aresuggested by the nature of nearby hydrogen bond partners: for example,Thr 275 O or Ser 277 N. See e.g. Tables in Appendix 2. For example, anyany negatively charged substituent would be compatible for interactingwith the amino acids of the polar pocket, including carboxylates,carbonyl, phosphonates, and sulfates, comprising 0 to 4 carbons. Anotherexample of an R₁ substitution is an oxamic acid that replaces the amineof the naturally occurring ligand with one or more carbonyl groups.

2) Incorporate hydrogen bond acceptor and donor groups into theR₁-substituent to provide broader interactions with the polar pocketscaffold. Such hydrogen bond acceptor and donor groups incorporated intothe R₁-substituent will allow interactions that would otherwise occurwith water molecules in the polar pocket. Specific waters include HOH504 (hydrogen bonds with Ala 225 O and Arg 262 NH); and HOH 503 hydrogenbonds with Asn 179 OD1, Ala 180 N), both of which are present in allfour complexes (TR LBD complexed with T3, TR LBD complexed with IpBr₂,TR LBD complexed with Dimit and TR LBD complexed with Triac). Analysisof the hydrogen bonding network in the polar pocket suggests replacementof HOH 504 with a hydrogen bond acceptor, and HOH 503 with an hydrogenbond donor (although the chemical nature of asparagine probably permitsflexibility at this site). Thus, incorporating a hydrogen bond acceptorin an R1 substituent that could take the place of the HOH504 orincorporating a hydrogen bond acceptor in an R1 substituent that couldpositionally replace the HOH503, or a combination thereof, are methodsof designing novel synthetic TR ligands.

These two design approaches can be used separately or in combination todesign synthetic ligands, including those in Table 5 (below).

A corollary to this approach is to design specific interactions to theresidues Arg262 and Asn 179. The goal is to build in interactions tothese residues by designing ligands that have R₁ substituents that formhydrogen bonds with water molecules or charged residues in the polarpocket.

High-affinity ligands also may be designed and selected using smallmolecules that bind to proximal subsites of the target nuclear hormonereceptor that are identified in a structure-based screen and then linkedtogether in their experimentally determined bound orientiations. Such amethod has been described in design of high-affinity ligands for theFK506 binding protein (FKBP), stromelysin, gelatinase A, and humanpapillomavirus E2 (Hajduk et al., Science 278:497-499 (1997)), whichreference and its references are incorporated herein by reference. Thepreferred small molecules for screening are compounds of Formula I orderivatives thereof. For example, a compound of Formula I (φ-X-φ) or aderivative thereof (φ-X or X-φ) is screened for binding a target nuclearhormone receptor LBD. Proximal subsites of the nuclear hormone receptorinclude the hydrophobic and polar pockets of the LBD, and substitesextended therefrom. As an example, Fourier transformation or nuclearmagnetic resonance (NMR)-based structure screens can be used. When aNMR-based screen is used, binding can be detected from the amidechemical shift changes observed in two-dimensional heteronuclear singlequantum correlation (HSQC) spectra aquired in the presence and absenceof added compound. Once two ligands are identified that bind to thereceptor, the crystal or solution structure of the ternary complex isdetermined. From the structural information, a compound is synthesizedwhich links the two ligands, where the linker is selected based onstructural information. The new compound is then screened for bindingaffinity, for example, using a binding assay as described herein. Only afew linked ligands need to synthesized and screened when using thisapproach.

Compounds of the invention also may be interatively designed fromstructural information of the compounds described above using otherstructure-based design/modeling techniques (Jackson, R. C.,Contributions of protein structure-based drug design to cancerchemotherapy. Semninars in Oncology, 1997, 24(2)L16172; and Jones, T.R., et al., J. Med. Chem., 1996 39(4):904-917). TABLE 5 Synthetic TRLigands

R1 R2 R3 R5 R6 X R′2 R′3 R′4 R′5 R′6 CO2H H Me Me H O H Me OH Me HCH2CO2H I I S Et SH Et CH2CH2CO2H Br Br nPr NH2 nPr CH2CH(NH2)CO2H Cl CliPr iPr OCH2CO2H Et Et Ph nBu OCH2CH2CO2H OH OH I nPen NHCH2CO2H NH2 NH2Br nHex NHCH2CH2CO2H SH SH Cl Ph CH2COCOCO2H hetero cycle NHCOCOCO2Haryl COCO2H CF2CO2H COCH2CO2H

Any combination of the above substituents in the biphenyl ether scaffoldstructure shown above may result in a potentially pharmacologicallyuseful ligand for the thyroid hormone receptor. These novel ligands maybe antagonists of the thyroid receptor. TABLE 6 TR-α LBD-122/410 DimitT3 IpBr₂ Triac Data collection Cell dimensions a (Å) 117.16 117.19117.18 118.19 b (Å) 80.52 80.20 80.12 81.37 c (Å) 63.21 63.23 63.1363.73 β (°) 120.58 120.60 120.69 121.00 Resolution (Å) 2.2 2.0 2.1 2.45Obs. Reflections, 57031 64424 66877 83573 (no.) Unique 22327 21023 2396618453 Reflections, (no.) Completeness, 87.0 82.4 93.7 96.0 (%) *R_(sym)(%) 3.9 3.5 4.5 7.5 Phasing (15.0-2.5 Å) †R_(der) (%) — 19.6 11.6 No. ofsites — 3 2 ‡Occupancy — 44.6 (19.8) 35.0 (Anomalous) — 50.2 (23.7) 35.039.2 (22.3) §F_(H)/E centric (acentric) 15.0-5.0 Å — 3.67 (4.61) 2.25(3.09)  5.0-3.0 Å — 2.23 (2.75) 1.25 (1.85)  3.0-2.5 Å — 1.64 (1.99)1.15 (1.57) ∥R_(Cullis) (%) 15.0-5.0 Å — 33 44  5.0-3.0 Å — 45 63 3.0-2.5 Å — 60 65 Mean figure of 0.62 — — merit MR Phasing (10-3.5 Å)Rotation Search: ⊖₁ = 309.37 Euler Angles (°) ⊖₂ = 48.96  ⊖₃ = 127.28 §correlation 34.3 coefficient Translation x = 0.1571 Search: Fractional y= 0.000  coordinates z = 0.3421 § correlation 65.8 Coefficient R factor31.2 Refinement 15.0-2.2 5.0-2.0 15.0-2.2 25-2.5 Resolution (Å)¶R_(cryst (%)) 20.5 22.1 21.4 23.6 R_(free (%)) 22.7 24.0 22.4 24.1

TABLE 7 TR-β LBD-202/461 Triac T3 GC1 Data collection Space Group P3121Cell dimensions a (Å) 68.9 68.45 68.73 c (Å) 131.5 130.56 130.09Resolution (Å) 2.4 3.1 2.8 Obs. Reflections, (no.) 80196 55103 54104Unique Reflections, (no.) 14277 6847 8987 Coverage (%) 97.0 95.7 97.1*R_(sym) (%) 5.1 4.6 5.5 MR Phasing (15.0-3.5 Å) Rotation Search ⊖₁ =39.13 Euler Angles (°) ⊖₂ = 68.00 ⊖₃ = 323.6 § correlation coefficient21.6 (Highest false peak) (10.8) Translation Search x = 0.748 FractionalCoordinates y = 0.158 z = 0.167 § correlation coefficient 57.5 (Highestfalse peak) (38.7) 0.612 *R factor 40.7 40.8 Refinement 30-2.4 30-2.9Resolution (Å) ¶R_(cryst (%)) 25.3 27.3 R_(free (%)) 28.9 33.4

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. The nuclearreceptor ligands, particularly the TR ligands, of these references areherein incorporated by reference and can be optionally excluded from theclaimed compounds with a proviso.

Headings and subheadings are presented only for the convenience of thereader and should not be used to construe the meaning of terms usedwithin such headings and subheadings.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

APPENDIX 1

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Zenke, M., Munoz, A., Sap, J., Vennstrom, B. & Beug, H. Cell 61,1035-1049 (1990). LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00001 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00002 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00003 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00004 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00005 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00006 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00007 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00008 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00009 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00010 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00011 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00012 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00013 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00014 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00015 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00016 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00017 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070027215A1-20070201-T00018 Please refer to the end of thespecification for access instructions. LENGTHY TABLE The patentapplication contains a lengthy table section. A copy of the table isavailable in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070027215A1)An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1-60. (canceled)
 61. A method of using a model of a nuclear hormonereceptor, or ligand binding domain thereof, bound to a nuclear hormonereceptor ligand, the method comprising: providing structural informationcorresponding to an atomic coordinate model of the nuclear hormonereceptor, or ligand binding domain thereof, bound to the nuclear hormonereceptor ligand; and, accessing the structural information.
 62. A methodof determining whether a potential nuclear ligand is likely to bind to anuclear hormone receptor ligand binding domain, the method comprising:accessing structural information corresponding to an atomic coordinatemodel of the nuclear hormone receptor ligand binding domain; accessingstructural information corresponding to the ligand; and, modelingbinding of the potential ligand to the nuclear hormone receptor ligandbinding domain, thereby determining whether the potential ligand islikely to bind to the nuclear receptor ligand binding domain.
 63. Themethod of claim 62, wherein modeling binding of the potential ligandcomprises modeling whether the ligand binding domain folds around thepotential ligand to form a buried ligand binding cavity.
 64. The methodof claim 61 or 62, wherein, in the atomic coordinate model, the nuclearreceptor folds around the ligand or potential ligand to form a buriedligand binding cavity.
 65. The method of claim 61 or 62, wherein theatomic coordinate model of the nuclear hormone receptor ligand bindingdomain comprises structural information for a bound ligand.
 66. Themethod of claim 61 or 62, wherein the structural information comprisesinformation corresponding to data from Appendix 3, 4, 5, 6, 7 or
 8. 67.The method of claim 61 or 62, wherein accessing the structuralinformation comprises performing a Forrier transform of crystallograpicdata corresponding to the nuclear receptor, the nuclear receptor ligandbinding domain, or the nuclear receptor bound to the ligand at thenuclear receptor ligand binding domain.
 68. The method of claim 61 or62, comprising modeling which amino acid or amino acids of the nuclearreceptor or nuclear receptor ligand binding domain interact with atleast a first chemical moeity of the ligand.
 69. The method of claim 68,further comprising designing a modified ligand, which modified ligand isselected to increase or decrease a modeled interaction between the aminoacid or amino acids and the first chemical moiety.
 70. The method ofclaim 61 or 62, comprising crystalizing the nuclear hormone receptorbound to the nuclear receptor ligand, wherein the structural informationis derived from a crystal structure of the resulting crystal.
 71. Themethod of claim 61 or 62, wherein the ligand is a computationallydesigned ligand.
 72. The method of claim 61 or 62, wherein the ligand isa compound of Formula
 1. 73. The method of claim 61 or 62, wherein thenuclear hormone receptor is a TR receptor.
 74. The method of claim 61 or62, wherein the nuclear hormone receptor is selected from the groupconsisting of: a glucocorticoid receptor, an androgen receptor, aprogestin receptor, an estrogen receptor, a vitamin D receptor, aretinoid receptor, an icosanoid receptor, and a peroxisome receptor. 75.A method for identifying a compound capable of selectively modulatingthe activity of a thyroid hormone receptor (TR) isoform, said methodcomprising: modeling test compounds that fit spatially andpreferentially into a TR ligand binding domain (TR LBD) isoform ofinterest using an atomic structural model of a TR LBD isoform bound to atest compound, wherein said atomic structural model employs highresolution structural information corresponding to an atomic coordinatemodel of the thyroid hormone receptor, or ligand binding domain thereof,bound to the thyroid hormone receptor ligand, screening said testcompounds in a biological assay for TR isoform activity characterized bybinding of a test compound to a TR LBD isoform, and identifying a testcompound that selectively modulates the activity of a TR isoform. 76.The method of claim 75, wherein said atomic structural model employshigh resolution structural information corresponding to data fromAppendix 3, 4, 5, 6, 7 or
 8. 77. A method for identifying a thyroidhormone receptor (TR) agonist or antagonist ligand, said methodcomprising the steps of: providing the atomic coordinates of a TR ligandbinding domain (TR LBD) to a computerized modeling system, wherein saidatomic coordinates are based on high resolution structural informationcorresponding to an atomic coordinate model of the thyroid hormonereceptor, or ligand binding domain thereof, bound to the thyroid hormonereceptor ligand; modeling ligands which fit spatially into the TR LBD;and identifying in a biological assay for TR activity a ligand whichincreases or decreases the activity of said TR, whereby a TR agonist orantagonist is identified.
 78. The method of claim 77, wherein saidatomic coordinates are based on data from Appendix 3, 4, 5, 6, 7 or 8.79. A method of identifying a compound that selectively modulates anactivity of a thyroid hormone receptor (TR) compared to other nuclearhormone receptors, said method comprising: modeling compounds which fitspatially into a TR ligand binding domain (TR LBD) using an atomicstructural model of a TR LBD, wherein said atomic structural modelemploys high resolution structural information corresponding to anatomic coordinate model of the thyroid hormone receptor, or ligandbinding domain thereof, bound to the thyroid hormone receptor ligand;selecting a compound comprising conformationally constrained structuralfeatures that interact with conformationally constrained residues of aTR LBD; and, identifying in a biological assay for TR activity acompound that selectively binds to a TR LBD compared to other nuclearreceptors, whereby a compound that selectively modulates a TR isidentified.
 80. The method of claim 79, wherein said atomic structuralmodel employs high resolution structural information corresponding todata from Appendix 3, 4, 5, 6, 7 or
 8. 81. A method for identifying athyroid hormone receptor (TR) agonist or antagonist ligand thatselectively modulates an activity of a TR compared to other nuclearreceptors, said method comprising the steps of: providing the atomiccoordinates of a TR ligand binding domain (TR LBD) to a computerizedmodeling system, wherein said atomic coordinates are based on highresolution structural information corresponding to an atomic coordinatemodel of the thyroid hormone receptor, or ligand binding domain thereof,bound to the thyroid hormone receptor ligand; modeling ligands which fitspatially into the TR LBD and which interact with conformationallyconstrained residues of a TR LBD conserved among TR isoforms; and,identifying in a biological assay for TR activity a ligand whichselectively binds to said TR and increases or decreases the activity ofsaid TR, whereby a TR agonist or antagonist that selectively modulatesthe activity of a TR is identified.
 82. The method of claim 81, whereinsaid atomic coordinates are based on data from Appendix 3, 4, 5, 6, 7 or8.